Current Biotechnology

Current Situation and Prospect of Garlic Industry Development in China
Zitian WANG, Shuo FAN, Rongshan LIN, Yaming XIE, Hailei WEI, Miao GAO, Min LIN
2025, 15(2): 179-188. DOI: 10.19586/j.2095-2341.2024.0173
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As the world's largest garlic producer， China dominates global cultivation area and output while possessing abundant garlic resources. By 2023， the country's garlic cultivation had reached 845 600 hm² with an annual production of 20.798 5 million tonnes， establishing a robust foundation for industrial development. Garlic serves not only as an essential culinary seasoning， but also finds extensive applications across pharmaceutical， healthcare， and food processing sectors. Despite its substantial industrial scale， economic returns remain concentrated in raw material supply and primary processing stages. The development of deep-processing technologies and value-added products lags significantly， creating a "high quantity， low value-added" paradox within the industrial chain. This paper systematically analyzed garlic's economic value， varietal distribution， spatial cultivation patterns， and industrialization status， identifying key challenges in industrial upgrading. It proposed strategic pathways for enhancing product value addition and promoting cross-chain integration. Through constructing a deep-processing and industrial integration model， this research provided both theoretical guidance and practical strategies for sustainable development of China's garlic industry， ultimately aiming to drive industrial upgrading and strengthen global market competitiveness.

Advances on Phage Genetic Engineering Technology and Applications
Yaoxuan JIAO, Min LI, Xinya QU, Tianling LIU, Xiumei SHENG, Xiaoxiang ZHOU
2025, 15(2): 189-200. DOI: 10.19586/j.2095-2341.2024.0138
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Antibiotic resistance has become one of the most critical threats to global health. The emergence of multi-drug resistant bacterial infections has led to increasingly high morbidity and mortality rates across medical， industrial， agricultural， and ecological domains. Phages can specifically lyse multi-drug resistant pathogens. However， due to their narrow host range， the presence of unfavorable genes in their genomes， and other limitations， only a limited number of phages have been successfully applied to combat multidrug-resistant bacterial infections. With editable and efficient features， phage genetic engineering provides a promising approach for expanding phage host ranges and designing "safe， green， and efficient" novel phages. This review systematically summarized recent advances in phage genetic engineering technologies while highlighting their practical applications in clinical therapies against drug-resistant infections， agricultural production， and ecological remediation. These insights established theoretical foundations for phage modification and their effective utilization in diverse fields.

Knowledge Gaps and Chanllenges in Microbial Fermentation of Traditional Chinese Medicine: From Strain Selection to Quality Control
Xiaoqi WU, Wenjing GONG, Guoyu LI, Ang LI, Jihua WANG, Di CUI
2025, 15(2): 201-211. DOI: 10.19586/j.2095-2341.2024.0205
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Microbial fermentation of traditional Chinese medicinal materials serves as a pivotal technique for novel drug discovery in traditional Chinese medicine （TCM） processing. While contemporary biotechnology advancements have progressively clarified the mechanisms and processes underlying TCM fermentation， persisting challenges include the selection of appropriate microbial strains， elucidation of fermentation mechanisms， determination of relationships between fermentation endpoints and quality control markers， and standardization of fermentation protocols. This review systematically summarized the specific microbial mechanisms involved in TCM fermentation and evaluated current research through three critical dimensions： functional diversity of fermentative microorganisms， biochemical mechanisms of fermentation， and process optimization strategies. Furthermore， it prospected future developmental trajectories in this field. The review aimed to provide theoretical foundations and technological references for defining fermentation endpoints， establishing quality standards， optimizing standardized processes， and advancing drug innovation within the TCM industry chain.

Immunogenicity Formation Mechanism and Control Strategy of Biopharmaceuticals
Zhaohui CUI, Ling GUO, Xudong SHEN, Yi LIN, Lili ZHAI
2025, 15(2): 212-219. DOI: 10.19586/j.2095-2341.2024.0175
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Biopharmaceuticals have achieved increasingly significant therapeutic effects in the treatment of tumors， autoimmune diseases and other complex diseases. However， there is a risk of immunogenicity when using biopharmaceuticals for treatment， which can reduce drug efficacy and affect treatment outcomes， even cause severe adverse reactions. Reducing or eliminating the immunogenicity of biopharmaceuticals on the basis of maintaining their pharmacokinetic properties and therapeutic efficacy has become an important aspect of the drug development process. Understanding the complex mechanisms driving the immunogenicity of biopharmaceuticals and developing effective strategies to reduce immunogenicity risks are crucial for improving drug efficacy and safety. The article reviewed the development of mechanism of immunogenicity in biopharmaceuticals， discussed the factors that affected immunogenicity and focused on strategies used to reduce immunogenicity in drug development， in order to provide a reference for the research and development of biopharmaceuticals.

Research Progress on Neuroprotective Effects of Betaine
Tianyuan ZHAO, Jing WANG, Yulu WANG, Chunsen YUAN, Xuechai CHEN
2025, 15(2): 220-225. DOI: 10.19586/j.2095-2341.2024.0163
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As a naturally occurring compound， betaine has attracted widespread attention in the field of neuroprotection in recent years. The unique biochemical properties and multiple functions of betaine make it has potential application value in the treatment of neurodegenerative diseases， neuroinflammation and nerve injury. Betaine has the functions of promoting the metabolism of homocysteine， reducing the level of oxidative stress， regulating the expression of γ-aminobutyric acid， etc. It has a therapeutic role in various neurological diseases such as Alzheimer's disease， depression， and Parkinson's disease， with relatively minor side effects. However， current research on the neuroprotective effects of betaine is still in its infancy， and further exploration is needed to understand its mechanism of action and optimal application scenarios. The article reviewed the research progress on the neuro protective effects of betaine， aiming to provide new ideas and methods for the treatment of neurological diseases.

Anti-tumor Effects of Gentian Violet
Geyemuri WU, Jianqiang WU
2025, 15(2): 226-233. DOI: 10.19586/j.2095-2341.2025.0011
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Malignant tumors remain a significant challenge in global public health， posing severe threats to human health. While modern medicine has achieved remarkable advances in anticancer therapies， the development of novel antitumor drugs still faces challenges including high costs， long development cycles， and limited accessibility， particularly in resource-limited regions. Therefore， exploring cost-effective and potent antitumor agents is of paramount importance. Gentian violet （GV）， a traditional triarylmethane dye， has been widely used historically for tissue staining， antibacterial， and antifungal treatments. Recent studies have revealed that GV exhibits prominent antitumor activity through the modulation of multiple cellular signaling pathways. Notably， GV demonstrates inhibitory effects against diverse malignancies， including lymphoma， hepatocellular carcinoma， melanoma， breast cancer， ovarian cancer， colorectal cancer， and glioma. Owing to its advantages such as low drug resistance， minimal toxicity， affordability， and reduced regulatory barriers， GV has emerged as a promising candidate for drug repurposing strategies in oncology. This review comprehensively examined the antitumor effects of GV and its underlying molecular mechanisms， providing a scientific foundation for future mechanistic investigations and clinical translation.

Clinical Progress in Targeted Therapy and Immunotherapy in Breast Cancer
Yuqin BIAN, Keran DONG, Junzi LU, Enming ZHONG, Wanying GU, Jingshu ZHAO, Hongshu SUI
2025, 15(2): 234-240. DOI: 10.19586/j.2095-2341.2024.0165
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Breast cancer is the most common malignant tumor in women all over the world， and its heterogeneity brings great challenges to treatment. The progress of targeted therapy and immunotherapy brings new hope for the treatment of breast cancer. Targeted therapy inhibits tumor progression by inhibiting specific targets. Potential therapeutic targets for breast cancer include serine/threonine kinases （AKT）?， cyclin-dependent kinases 4 and 6 （CDK4/6）?， polyadenosine diphosphoribose polymerase （PARP）， and various growth factors. Methods of immunotherapy include immune checkpoint blocking， vaccination， and adoptive T cell therapy. The review summarized the clinical progress of targeted therapy and immunotherapy for breast cancer， aiming to providing reference for breast cancer related research and disease treatment.

The Role and Research Progress of Gene P53 in Osteosarcoma
Yanjie WANG, Bo QIU
2025, 15(2): 241-246. DOI: 10.19586/j.2095-2341.2024.0132
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Osteosarcoma （OS） is the most common primary bone malignant tumor，with strong local invasion， metastasis， and a high risk of recurrence， which mainly affects the health of children and adolescents. At present， chemotherapy and surgery are the main methods for treating OS， but the efficacy is not significant， and immune checkpoint inhibitors have poor clinical application efficacy， requiring new therapeutic targets. As a classic tumor suppressor gene， P53 gene participates in many biological processes such as cell cycle regulation， DNA repair and cell death. The mutation of P53 is widespread in OS and closely related to tumor aggressiveness and prognosis. In addition to its traditional role in apoptosis， P53 also plays an important role in non-classical cell death pathways such as autophagy and ferroptosis， which makes it a key target for OS treatment. This article reviewed the role of P53 in osteosarcoma， with a focus on exploring the regulatory mechanisms of P53 in apoptosis， autophagy and ferroptosis， as well as its research progress in OS. The article aimed to provide new theoretical basis and guidance for future therapeutic targets and strategies for OS.

Research Progress on the Role of Toll-like Receptor 7 in Cardiovascular Diseases
Ruoliu ZHANG, Mingwei BAO
2025, 15(2): 247-253. DOI: 10.19586/j.2095-2341.2024.0152
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Cardiovascular disease is a type of disease with high incidence, disability, and mortality rates, which seriously threatens human health and imposes a huge economic and health burden on society. At present, there are countless studies on cardiovascular diseases, but the pathogenesis is still unclear. The involvement of TLRs in the occurrence and development of chronic inflammation in cardiovascular disease has been supported by experimental and preclinical data. Toll like receptor 7 (TLR7), as a key member of TLRs, can recognize bacterial, viral sources, and its own nucleic acids, and activate inflammatory responses through the myeloid differentiation factor 88 (MyD88) pathway to mediate immune responses. This review summarized the research progress of TLR7 in cardiovascular diseases such as atherosclerosis, myocarditis and cardiomyopathy, hypertension and pulmonary hypertension, and its potential as a drug target for cardiovascular diseases, in order to provide new ideas for clinical treatment of diseases.

Research Progress on Pathogenic Mechanisms and Treatment Strategy of Coronary Artery Disease
Hao LIU, Xiang LI
2025, 15(2): 254-262. DOI: 10.19586/j.2095-2341.2024.0151
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Coronary artery disease （CAD） remains the leading cause of global mortality. This article reviewed the pathophysiology of atherosclerosis， highlighting the contributions of genetic predisposition， inflammatory immune mechanisms， and dyslipidemia to disease pathogenesis. We systematically analyzed modifiable and non-modifiable risk factors driving disease progression， while discussing recent advances in preventive strategies and therapeutic interventions. By synthesizing current knowledge of critical pathogenic pathways， this work established a conceptual framework for developing innovative approaches to combat atherosclerosis.

Joint Transcriptome Analysis of Maize Under Salt Stress and MeJA Treatment
Zhuoying LIU, Xiaojin ZHOU, Yanli HUANG, Sen PANG
2025, 15(2): 263-275. DOI: 10.19586/j.2095-2341.2024.0183
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Maize， an important food and feed crop， faces severe growth inhibition， yield reduction， and quality deterioration under salt stress. Jasmonic acid and its derivatives （JAs）， crucial phytohormones involved in plant defense mechanisms， have been shown through studies in model plants to play essential roles in salt stress responses. To investigate JAs-mediated salt stress adaptation mechanisms in maize， we subjected seedlings to combined treatments of 200 mmol·L^-1 NaCl and 100 μmol·L^-1 methyl jasmonate （MeJA） for six hours. Transcriptomic analysis of shoots and roots identified differentially expressed genes （DEGs） associated with both JA signaling and salt stress response. Eight overlapping DEGs from shoot-root comparisons were subsequently validated using RT-qPCR. The study revealed 362 and 803 stress-responsive DEGs in overground and subterranean tissues， respectively. Functional enrichment analyses （GO and KEGG） demonstrated these genes participate in carbohydrate metabolism/transport， defensive secondary metabolite biosynthesis， antioxidant enzyme production， along with abscisic acid and ethylene signaling pathways. These findings indicated that JA signaling activates specific genetic and metabolic networks underlying salt stress adaptation of maize， providing critical insights for further elucidating the molecular mechanisms of JA-mediated salt tolerance regulation.

Research of Detection Method for Genetically Modified Herbicide-tolerant Soyabean LP012-1 Transformant by Quantitative Real-time PCR
Yueqiu ZHANG, Fangqi FU, Hua ZHANG, Yifan CHEN, Chenyao WANG, Hongye JIANG, Yahui LI, Yichen LIAO, Dan WANG, Yu SUN, Wei FU, Hong CHEN
2025, 15(2): 276-286. DOI: 10.19586/j.2095-2341.2024.0208
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The standardized genetically modified detection's method provides important technical support for the industrial application and quantitative identification system implementation of biological breeding. Based on the left boundary of the foreign insertion sequence， a quantitative real-time PCR （qPCR） detection method was established with the genetically modified soybean LP012-1 with genetically modified herbicide-tolerant soyabean selected as the research object. The target sequence of the left boundary of the foreign insertion sequence was designed as the primer and its corresponding probe， and the optimal primer combination was selected by screening. The primer and probe concentrations and annealing temperature were optimized， and the linear dynamic range was determined to be from 0.018 to 100 ng. The detection limit and the quantitative limit were 0.05% and 0.10%， respectively. The correctness， precision， robustness， and joint validation error between laboratories were less than 25%. The qPCR detection method established in this study has good specificity， high sensitivity， and strong robustness， which can provide technical support for the safety supervision of transgenic soybean LP012-1 and serve the commercialization of biological breeding.

Intelligent Mining of Novel GH45 Cellulases and its Efficient Expression in Trichoderma reesei
Jin WANG, Jing BAI, Jing ZHAO, Jian TIAN, Wei ZHANG, Yuan WANG, Haomeng YANG, Meishan SHI, Beibei ZHANG, Xinxin XU, Huoqing HUANG
2025, 15(2): 287-295. DOI: 10.19586/j.2095-2341.2024.0189
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Utilizing bioinformatics methods and protein property prediction models to mine novel GH45 family cellulases， and achieving their efficient expression in Trichoderma reesei， in order to provide a new enzyme source for animal feed processing. By using the Preoptem protein model prediction tool combined with the public database Uniparc， the GH45 family cellulase TpCel45A from Thielaviopsispunctulate was screened. We synthesized the encoded gene and construct an expression vector， and transformed it into a strain of Trichoderma reesei using protoplasts. The recombinant TpCel45A protein was obtained through expression and purification， and its enzymatic properties were determined. The research showed that the optimal temperature and pH for recombinant TpCel45A were 55 ℃ and 5.5， respectively， and exhibited good thermal stability. After 15 minutes of treatment at 80 ℃， the enzyme activity remained over 84%. In addition， TpCel45A exhibits good stability within the pH range of 4.0~9.0， with residual enzyme activity exceeding 52%. The determination of kinetic parameters showed that the K_m of the cellulase was 8.04 mg·mL^-1， V_maxis 19.04 mg·mL^-1·min^-1， the k_cat value is 377.1 s^-1， and the catalytic efficiency k_cat/K_m value is 46.9. The excavation， identification， and heterologous expression of TpCel45A provide a new enzyme source for the industrial application of cellulase. In addition， the excellent thermal stability and pH stability of TpCel45A make it have the potential for application in high-temperature granulation and different pH environments， and have important application value in fields such as feed proessing and biomass conversion utilization.

Reaction System Optimization and Application Potential Analysis of 4-Oxalocrotonate Tautomerase in Catalytic Synthesis of Cinnamaldehyde
Xi CHEN, Huoqing HUANG, Yingguo BAI, Yuan WANG, Chengqiang XIA, Huiying LUO, Bin YAO, Tao TU, Xiaoqing LIU
2025, 15(2): 296-304. DOI: 10.19586/j.2095-2341.2024.0209
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Cinnamaldehyde is an important natural compound widely used in the pharmaceutical， food， and feed additive industries. However， traditional chemical synthesis methods involve high energy consumption and environmental pollution， limiting their industrial application. Enzymatic catalysis， as a green synthesis pathway， offers high specificity and environmental advantages， making the exploration of enzymatic cinnamaldehyde synthesis highly significant. Using 4-OT to construct the pET-20b-4-OT expression vector， the 4-oxalocrotonate tautomerase （4-OT） was expressed and purified in Escherichia coli to verify its ability to catalyze cinnamaldehyde synthesis from benzaldehyde and acetaldehyde as substrates， and its performance under different pH， temperature， enzyme concentration， salt concentration， and substrate concentration conditions was evaluated. The high-performance liquid chromatography was used to measure cinnamaldehyde yield， and the effects of optimized conditions on enzyme activity and product formation were analyzed. The optimal pH and temperature for 4-OT were determined to be pH 7.3 and 37 ℃， respectively. At an enzyme concentration of 15 mg·mL^-1， cinnamaldehyde yield increased by 181% compared to the initial conditions. The K₂HPO₄-KH₂PO₄ buffer system and a salt concentration of 125 mmol·L^-1 significantly enhanced enzyme activity. Optimal substrate concentrations were determined to be 0.5 mmol·L^-1 for benzaldehyde and 500 mmol·L^-1 for acetaldehyde. Expanding the reaction system to 400 mL increased the cinnamaldehyde yield by 719-fold and the conversion rate by 143-fold. This study optimized the conditions for 4-OT-catalyzed cinnamaldehyde synthesis， significantly improving catalytic efficiency and yield， thus providing strong support for the industrial application of 4-OT in green cinnamaldehyde synthesis.

Screening and Condition Optimization of Endogenous Compound Microflora in Shale Oil Driven by Biological Fracturing Fluid
Siyuan QIU, Jingxue XU, Yiting ZHANG, Shengyang SUN, Dongxue SHENG, Jiaxin GAO, Lina QU
2025, 15(2): 305-313. DOI: 10.19586/j.2095-2341.2024.0143
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Hydraulic fracturing technology has unique advantages in the efficient extraction of shale oil， but the chemicals contained therein pose a high risk of polluting groundwater. By using the fermentation broth produced by shale oil endogenous functional bacteria as biological fracturing fluid， participating in the green exploitation of shale oil can significantly improve the recovery rate of shale oil and reduce the negative impact on the environment. The functional strains were screened by blood plates， and the yield of surfactant was used as the evaluation criterion， and the three functional strains were determined and the species information was identified by 16S rDNA sequencing. According to the different inoculation ratios of 1， 2 and 3 μL， the three strains were compounded into different strains of the same volume to obtain the optimal strain ratio， and the most suitable factors were screened out by single factor experiments， and then the orthogonal and response surfaces were further optimized to obtain high-yield culture conditions. The results showed that the three strains of high-efficiency functional bacteria were Pseudomonas， Bacillus and Taureella， with a combination ratio of 2∶2∶1. The optimal cultivation formula was lactose concentration 13.87 g·L^-1， ammonium persulfate 2.13 g·L^-1， iron sulfite 1.75 g·L^-1 and pH 6. Under these conditions， the surfactant yield of the strain was 315.51 mg·L^-1， an increase of 59.37% compared to the initial yield of 187.30 mg·L^-1. The results of this study can provide a reference for the development of biological fracturing fluid for shale oil.

Bibliometric Analysis of Mycotoxin Detection Technology Based on Web of Science Database: Visualization and Quantification
Zile WANG, Dongdong CHEN, Tao PENG
2025, 15(2): 314-324. DOI: 10.19586/j.2095-2341.2024.0161
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Mycotoxin contamination constitutes a significant public health concern that seriously threatens human health and livestock development， highlighting the crucial need to enhance detection capabilities. This study conducted quantitative analysis and visual representation of productive countries， institutions， researchers， publishing journals， highly cited documents， and keywords using data from the Web of Science Core Collection database. Our findings reveal that mycotoxin detection technology research is experiencing rapid growth， with an established international collaboration network. China leads in publication output and maintains world-class research status. Ghent University， Sarah De Saeger， and Food Control emerge as the most productive institution， researcher， and journal， respectively. Interdisciplinary integration has accelerated research progress， with Chinese scholars increasingly assuming leadership roles. Keyword clustering identifies four primary research themes： mycotoxin biosynthesis， instrumental analysis， immunoassay development， and toxicity/risk assessment. Recent high-frequency keywords in immunoassay research highlight emerging frontiers， particularly novel recognition elements （e.g.， aptamers） and advanced detection modalities （e.g.， biosensors） as current research hotspots.

Development of qPCR Standard Curves for Monitoring Population Dynamics in a Dual-bacterial Fermentative Hydrogen Production System
HanXiao MA
2025, 15(2): 325-332. DOI: 10.19586/j.2095-2341.2024.0133
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To better understand the interaction mechanism in hydrogen production by a dual-bacterial system comprising novel hydrogen-producing strains Bacillus cereus ATCC 14579 （T） and Brevundimonas naejangsanensis BIO-TAS2-2 （T）， and to effectively control the fermentation process for enhanced hydrogen yield， we established standard curves to monitor population dynamics of both strains. This study employed real-time quantitative PCR （qPCR） to develop a dynamic monitoring method. Hydrogenase genes of both strains were first amplified using specifically designed primers with whole genomes as templates （GenBank No.： CP015727.1 and CP015614.1）. The amplified hydrogenase sequences were then cloned into pMD19-T vectors to construct recombinant plasmids. These plasmid standards served as templates for qPCR amplification， enabling the establishment of standard curves through linear regression between log₁₀-transformed plasmid copy numbers and cycle threshold （C_t） values. The resulting calibration equations were y=-3.435 36x+53.569 87 （R2=0.997） for B. cereus and y=-3.374 62x+43.079 02 （R2=0.998） for B. naejangsanensis， with amplification efficiencies of 95.5% and 97.8%， respectively. The established standard curves meet the requirements， and they could be used to absolutely quantify two kinds of bacteria in fermentation system in subsequent studies. This methodology provides a foundation for investigating strain interactions during the mixed fermentation hydrogen production of both strains with different substrates and offers technical guidance for in vitro amplification of other enzymatic genes.

Study on the Key Technology for the Determination of Glycated Haemoglobin by Double-light POCT-type Latex Immunoturbidimetric Assay
Yanyan JIA, Hongzhuang GUO
2025, 15(2): 333-340. DOI: 10.19586/j.2095-2341.2024.0162
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Diabetes mellitus has become a common disease that endangers human health. Glycated haemoglobin is not affected by the concentration of blood glucose at the time of sampling， so its concentration detection is more meaningful for monitoring the disease. In this paper， based on Lambert's Beer's law， using the characteristic of selective absorption of light on substances， we designed a two-optical POCT-type latex immunoturbidimetric device for the determination of glycated haemoglobin， and carried out modelling simulation of its excitation optical path， incubation station and other main components， performance verification of the main performance of the instrument， and instrument comparison experiments with UV-1780 spectrophotometer. The experimental results showed that the linear correlation R²=0.998 4， the repeatability≤0.997 9%， the average heating rate of the incubation station was 0.44 ℃·s^-1， and the temperature uniformity was ≤±0.1 ℃ （the temperature was 37.3 ℃）. The trend was consistent with that of the UV-1780 spectrophotometer， which measured 21 different concentrations of glycated haemoglobin anticoagulant-positive QC samples， fully proving that the detection results of this device were accurate and reliable， and providing a pre-experimental basis for the POCT of the glycated haemoglobin detection device.

The Role of Ptbp1 in T Cell Acute Lymphoblastic Leukemia Mice
Xu ZHU, Yingnan ZHANG, Jinfa MA, Lihong SHI
2025, 15(2): 341-348. DOI: 10.19586/j.2095-2341.2024.0182
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The aim of this study is to explore the role of the classical RNA binding protein polypyrimidine bundle binding protein 1 （PTBP1） in the occurrence and development of T cell acute lymphoblastic leukemia （T-ALL）. Generating Mx1-Cre；Ptbp1^fl/fl mouse model that hematopoietic system specific knockout of Ptbp1 through Cre；Flox system. Mouse bone marrow lin^- cells were infected with Notch1 retrovirus， and the Ptbp1 knockout T-ALL model was established by transplantation experiments. The deletion of Ptbp1 was found to accelerate the disease progression of T-ALL， as indicated by a higher proportion of Notch1-GFP⁺ cells in the bone marrow of Ptbp1 knockout mice， suggesting faster disease progression. In addition， Ptbp1 knockout leukemia cells showed enhanced proliferation and decreased apoptosis， which was consistent with the rapid disease progression observed in mice. These results suggest the important role of Ptbp1 in the occurrence and development of T-ALL， and provide more understanding of Ptbp1 as a potential biomarker for T-ALL.

Influence of Different Lodging Sites on the Stemness of Leukemia Cells in a Mouse Leukemia Model
Shangshang WANG, Zhenya XUE, Haiyan XING, Xue YANG, Min WANG, Qing RAO
2025, 15(2): 349-354. DOI: 10.19586/j.2095-2341.2024.0177
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Self-renewal， quiescence， and resistance to chemotherapy are critical properties of leukemia stem cells， which contribute to disease relapse and poor prognosis after treatment. Bone marrow and spleen were identified as primary lodging sites for leukemia stem cells. Investigating the stemness properties of leukemia cells in these distinct microenvironments facilitated elucidation of the regulatory mechanisms underlying stemness maintenance. In this study， the AML1-ETO9a （AE9a） mouse leukemia model was used to evaluate the stemness of leukemia cells residing in bone marrow （endosteal region） and spleen. Stemness-related gene expression signatures were identified through RNA sequencing （RNA-seq） and gene set enrichment analysis （GSEA）. The proportion of cells in the G0 phase of the cell cycle was determined via DAPI/Ki67 labeling and flow cytometry， while colony-forming ability was assessed using a colony formation assay. Stemness-related molecules were validated by reverse transcription quantitative real-time PCR （RT-qPCR）. GSEA revealed that upregulated genes in endosteal leukemia cells， compared to spleen-resident leukemia cells， were significantly enriched in stemness-related gene sets， including quiescence. Leukemia cells in the endosteal region exhibited a higher proportion of G0-phase cells and enhanced colony-forming capacity. Genes highly expressed in endosteal leukemia cells showed significant enrichment in the interleukin-1 （IL-1） pathway. Specifically， IL-1β expression was markedly elevated in endosteal leukemia cells. These findings suggest that leukemia cells lodged in the endosteal region retain stemness properties， and the IL-1β-mediated IL-1 pathway may contribute to stemness maintenance within the bone marrow microenvironment. The IL-1 pathway represents a potential therapeutic target for acute myeloid leukemia （AML）.

Establishment of Stable Gastric Cancer Cell Model with PDE12 Knockdown and Overexpression Mediated by Lentivirus and its Cellular Localization Analysis
Yijin LIU, Wenyang XU, Jinji WEI, Huanxi SONG, Jiahui LI, Lina ZHANG
2025, 15(2): 355-363. DOI: 10.19586/j.2095-2341.2024.0156
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Phosphodiesterase 12 （PDE12） is a member of endonuclease/exonuclease/phosphatase （EEP） superfamily. To further investigate the function of PDE12 gene in cells， lentiviral infection technology was used to stably knock down and overexpress the PDE12 in gastric cancer cells and obtain the stable cell models. Firstly， the shRNA sequence targeting the knockdown of PDE12 gene was inserted into the lentiviral interference vector pLVX-shRNA2-Puro， and the full-length cDNA of PDE12 gene was inserted into the lentiviral overexpression vector pCDH-CMV-MCS-EF1-GFP-Puro. After comparing and identifying the sequencing results correctly， the lentivirus was packaged and infected human gastric cancer cell lines MKN-45 and BGC-823. Subsequently， stable cell lines were screened using puromycin， and the knockdown and overexpression efficiency of PDE12 at mRNA and protein levels in stable gastric cancer cell lines were verified by quantitative real-time PCR（qPCR） and Western blot， respectively. In addition， we also constructed a pEGFP-PDE12 eukaryotic expression vector and transfected it into MKN-45 and BGC-823 gastric cancer cells to detect the localization of PDE12 in gastric cancer cells by immunofluorescence analysis. The experimental results indicated that PDE12 expression in MKN-45-shPDE12 and BGC-823-shPDE12 stable cells was significantly reduced， while the expression level of PDE12 in MKN-45-PDE12 and BGC-823-PDE12 stable cells was dramatically increased， suggesting that the successful construction of MKN-45 and BGC-823 stable gastric cancer cell lines with PDE12 knockdown and overexpression. The observation results of confocal microscopy indicated that the green fluorescence expressed by PDE12 was distributed in both the nucleus and cytoplasm. Further mitochondrial staining analysis using MitoTracker Red revealed that the green fluorescence of PDE12 expression was co-localized with the mitochondrial MitoTracker Red fluorescence， suggesting that PDE12 is localized in the mitochondrial of gastric cancer cells. Overall， these research results provide a basis for further in-depth study of the function and mechanism of PDE12 in gastric cancer.

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