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Table of Content

    • Current Biotechnology
      Volume 9 Issue 3 25 May 2019
    • Review of Immunotherapy for Glioblastoma
    • LIU Mengyu1, XIE Fei1, ZHANG Xin1, ZHAO Pengxiang1,2*
    • 2019, 9(3):  223-230.  DOI: 10.19586/j.2095-2341.2019.0034
    • Abstract ( ) PDF (1052KB)( )
      • Immunotherapy aims to increase the bodys immune response to tumors. The primary means used today include increasing the immune response in the body part or generating a corresponding immune response against the targeted antigen stimulating organism. Among them, the latter usually introduces cells that restart the immune response into the body to directly act on the target antigen by the immune system. Clinically, immunotherapy is mainly applied to the treatment of solid tumors such as melanoma and prostate tumor. The central nervous system was once thought to be an immune privilege zone, but in recent years a large number of studies have shown that the brains immune system is at a high level of activation and can interact with brain tumors. Therefore, research and use of this immune function is very important for the development of treatment of malignant brain tumors. This article summarized recent advances in immunotherapy for malignant glioblastoma, and provided a detailed introduction to the immunological mechanisms of central nervous system and glioblastoma, immunotherapy vaccines, and T cell therapy in cancer therapy.
    • Advances on the Analysis Methods of N-glycosylation
    • ZHANG Xiaoqian, ZHANG Tengteng, YAN Pan, YANG Bichao, AN Fengxia*
    • 2019, 9(3):  246-252.  DOI: 10.19586/j.2095-2341.2018.0121
    • Abstract ( ) PDF (1830KB)( )
      • Glycosylation is one of the most common forms of post-translational modifications in eukaryotic proteins. As the main glycosylation type of glycoproteins, N-glycosylation plays an important role in many life activities. It has positive practical significance for qualitative and quantitative research of N-glycans. The conventional analysis strategy of N-glycosylation is to separate the N-glycans from the peptide chain by enzymatic digestion. But it is difficult to separate and analyze the N-glycans by classical reversed-phase chromatography and ultraviolet detection, because of the N-glycans is a polyhydroxyaldehyde compound. In response to this situation, scientists mostly use pre-column derivatization to make the sugar chains with ultraviolet or fluorescent groups, so as to increase the sensitivity of their detection. In this paper, the methods of separation and analysis for dissociative N-glycans were summarized in recent years. Among them, CE, HPLC and MS have great advantages in the separation and analysis of N-glycans, which are of great significance for the qualitative, quantitative and structural characterization of N-glycans in glycoproteins. Meanwhile, stable and reliable methods for N-glycans release and enrichment should be further developed, combined with modern efficient analytical methods and targeted measures for N-glycans analysis, which was expected to provide a basis for the analysis of N-glycosylation with high flux and accuracy.
    • Anti-tumor Effect of T Cells Mediated by Humanized Mesothelin Chimeric Antigen Receptor
    • LIU Yuanyuan, NIU Qing, FENG Xiaoming*
    • 2019, 9(3):  253-261.  DOI: 10.19586/j.2095-2341.2019.0028
    • Abstract ( ) PDF (7823KB)( )
      • Despite that CAR-T cell (chimeric antigen receptor-modified T cell) therapy has achieved remarkable outcomes in the treatment of hematological malignancies, there are still lots of obstructions in the treatment of solid tumors. Mesothelin is a tumor associated antigen over-expressing on tumor cell surface such as mesothelioma, ovarian, pancreatic. Most of the current studies about CAR-T cells targeting Mesothelin use scFv (single-chain variable fragment) of mouse are to build expression vector by gene editing, which shows some shortcomings in simulating clinical treatment and obstacles of translation of research to clinical treatment, so it is very necessary to develop humanized study. We constructed a CAR vector with a humanized anti-mesothelin scFv called P4, packaged the lenti-virus and used them to transfect human peripheral blood CD3+ T cells. We successfully produced P4 CAR-T cells with high and steady transfection efficiency. PCR results indicated that Mesothelin was expressed in human cervical cancer tumor cell line HeLa. In vitro experiments demonstrated that our P4 CAR-T cells could effectively kill HeLa cells. In vivo experiments showed that infusion of P4 CAR-T cells slowed the growth of established tumors slightly and prolonged the survival of tumor-bearing mice in comparison to the control group. Here we reported that the P4 CAR-T cell can be used as a potential therapeutic strategy against Mesothelin positive tumor, and it provided a basis for further research on synergistic strategies to improve the therapeutic effect in vivo and clinical treatment.
    • Comparative Study on Transduction of Lentivirus Packaged with Different Transfection Methods in Human Leukemia Cells
    • ZHANG Mingying1, YUAN Jiajia1, ZHANG Xiaoru1, XING Wen1, BAI Jie2, ZHOU Yuan1*
    • 2019, 9(3):  262-270.  DOI: 10.19586/j.2095-2341.2019.0016
    • Abstract ( ) PDF (11005KB)( )
      • Gene overexpression and knock down mediated by lentivirus is often used in leukemia research. There are some problems, such as high cost and low efficiency in suspension cell infection. In order to identify the effectiveness and safety of the lentivirus packaged with different transfection method in leukemia research, solving the problems of low infection efficiency and high cost during the lentivirus production, we packaged lentiviruses with linear polyethylenimine (LPEI) and liposomes, and then compared the infection ability and the effects on biological characteristics of leukemia cell lines of lentiviruses produced with these two methods. Using LPEI or liposomes to generate lentiviral particles, respectively, our studies showed that the titer of lentivirus packaged with LPEI was slightly higher than that packaged with liposomes under the same packaging conditions. Three leukemia cell lines (K562, HL60 and HEL) were infected with the same amount lentiviral particles according to multiplicity of cellular infection (MOI) and lentiviral titer, and there was no difference in infection efficiency between the two groups. The cell proliferation of leukemic cells infected with lentivirus produced with LPEI was slightly slower than that in liposome group, the effects on induced differentiation and colony forming ability of these two methods were similar. Therefore, using lentivirus produced with LPEI transfection method can be an economical and practical choice in experimental studies in leukemia.
    • Response of Grain Starch and Sucrose Metabolism-related Enzyme Activities to Night Temperature Rise in Conventional Indica Rice Huanghuazhan
    • ZHANG Wenqian, WANG Yaliang, ZHU Defeng, CHEN Huizhe, XIANG Jing, ZHANG Yikai, WU Hui, HU Guohui, YI Zihao, ZHANG Yuping*
    • 2019, 9(3):  283-289.  DOI: 10.19586/j.2095-2341.2019.0010
    • Abstract ( ) PDF (1602KB)( )
      • In order to reveal the influence law of night temperature rise on the physiological process of grain filling during the filling process, the temperature change in the natural environment was simulated by using the climate chamber, and three different night temperature treatments, 21℃ (T1), 25℃ (T2) and 29℃ (T3), were set up in the three stages of rice filling process, which were P1 (the first 10 d after rice flowering), P2 (11~20 d after rice flowering) and P3 (21~30 d after flowering), and in the meantime, the highest temperature of daytime is 32℃. The effects of night temperature rise in different filling periods on the contents of grain starch and sucrose and their metabolism-related enzyme activities were studied during rice filling. The results showed that the activities of starch and sucrose metabolism-related enzymes of rice grains in P1 and P2 periods after rice flowering were significantly affected by the increase of night temperature, among which P1 period was the most sensitive period for starch metabolism to the change of night temperature. At the early stage of filling（P1）, the content of starch increased significantly with the increase of night temperature, as well as the contents of amylose and amylopectin, while the content of sucrose decreased. Under the same condition, the activities of sucrose synthase, soluble starch synthase and starch branching enzyme decreased, and the activities of sucrose phosphate synthase increased during the daytime and decreased at night. This indicated that the increase of night temperature had a sustained effect on the enzyme activity, that was, the enzyme activity during daytime was also affected by the increase of night temperature. The results provided a reference for understanding the influence mechanism of climate change on grain material accumulation during rice grain filling.
    • Antioxidant Ability Analysis of All Fermented Cultures and Their Ingredients of Two Strains of Lactobacillus
    • SHO Takami, LI Li, CHEN Xindan, ZHANG Jing, XU Weina, XU Jianxiong*
    • 2019, 9(3):  290-295.  DOI: 10.19586/j.2095-2341.2018.0115
    • Abstract ( ) PDF (1331KB)( )
      • To study on the antioxidant ability of all fermented cultures and their ingredients of Lactobacillus plantarum CGMCC 1.557 and Lactobacillus casei CGMCC 1.570, all fermented cultures, fermented supernatants, cell suspensions and intracellular extracts of the two strains were prepared separately first. Then their scavenging rates of three free radicals (DPPH·, O2·-, ·OH) and the activity of three antioxidases, including total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), were detected to evaluate the antioxidant ability of all fermented cultures and their ingredients of the two strains. The results demonstrated that all fermented cultures of both L. plantarum and L. casei possessed good radical-scavenging ability and decent activity of T-SOD and GSH-Px. Compared with cell suspensions and intracellular extracts, fermented supernatants of both Lactobacillus had significantly higher scavenging rates of DPPH· and O2·-, and higher activity of T-SOD (P<0.05). Cell suspensions of both Lactobacillus had the highest scavenging rate of ·OH（P<0.05）. The study indicated that two strains, L. plantarum CGMCC 1.557 and L. casei CGMCC 1.570, possessed good antioxidant ability, which mainly relied on antioxidase activity of fermented supernatants and cell. The results of the research provided theoretical foundation for further development on antioxidant Lactobacillus products.
    • Improvement of Cotton Mitochondrial Gene cRT-PCR and its Application in Searching for CMS Related Genes
    • WANG Peilin, ZHOU Lili, LIANG Chengzhen, MENG Zhigang, GUO Sandui*, ZHANG Rui*
    • 2019, 9(3):  303-308.  DOI: 10.19586/j.2095-2341.2018.0134
    • Abstract ( ) PDF (1768KB)( )
      • Plant mitochondria is a type of organelles containing genetic material，which provides necessary energy for cells. In agricultural production, genovariation of mitochondrial gene may lead to cytoplasmic male sterility, providing a convenient female parent for hybird breeding. Despite successful utilization of CMS and restoration of fertility (Rf) in breeding programs, the underlying mechanisms of these processes remain elusive because the mitochondrial genome structure is complex, and occurrence of different types of CMS is often caused by specific mutations consequently limiting gene cloning. This study summarized the techniques to improve cRT-PCR technology and present detailed characteristics mitochondrial gene involved in CMS. After RNA ligase cyclizes the 5′ and 3′ ends of mitochondrial mRNA, random primers were used for reverse transcription to ensure the same integrity as specific primers. Based on the mitochondrial gene transcript, cRT-PCR was performed on all mitochondrial coding genes, which provided insight of structure of mitochondrial coding genes. Therefore, we used this system to explore cotton CMS genes, thus quickly figuring out abnormal factors and providing a new method for mitochondrial functional genomic analysis.
    • The Optimization of the Bradford Assay for Protein Quantitation in Proteomic Research
    • CUI Weitong, XUE Huaru, CHENG Hongda, ZHANG Haibin, WANG Qinglu*
    • 2019, 9(3):  309-315.  DOI: 10.19586/j.2095-2341.2018.0126
    • Abstract ( ) PDF (1234KB)( )
      • Bradford assay is the most widely used protein quantification method for its low cost, high sensitivity and rapid and simple determination. To enhance the suitability of the Bradford assay for proteomic research, the assay was optimized specifically for two-dimensional electrophoresis (2-DE) samples. Through constructing the correspondence of protein content (0~1 000 μg) with absorbance at 595 nm, the linearity range was determined at 0~100 μg. The interference of lysis buffer and its components, and different detergents was tested. Results showed that there was a positive correlation between interference of lysis buffer and sample volume. By analyzing the absorbance change at 595 nm of protein samples with or without lysis buffer, lysis buffer was found to have an effect on the stability of protein-dye complex and the proposed measurement time for 2-DE samples was between 3 and 30 min after mixing protein samples with dye reagent. Increasing in phosphoric acid concentration from 8.5% to 10.2% (w/V) in the dye reagent can improve the stability of protein-dye complex. As the absorbance of the same amount of protein was quite different whether or not lysis was present, the addition of corresponding volume of lysis buffer to the standard protein samples and the reagent blank must be ensured. Sample volume was adjusted from 100 μL to 10 μL. The adjustment of sample volume lowered fluctuation of measured data and improved linearity of the standard curve, as well as simplified the quantitative analysis by elimination of dilution process.

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