Application of TARP luciferase reporter system in function identification of CAR-T cells / 细胞与分子免疫学杂志

Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.

Research on the experimental design of medical molecular biology in the era of precision medicine / 中华医学教育探索杂志

Objective Through the design of comprehensive experiments, the precise medical philo-sophy was put into the medical molecular biology experimental teaching, and to explore its effect. Methods Eight-year medical students of Grade 2012 and Grade 2013 in the Fourth Military Medical University were chosen as the teaching subjects. Experimental group consisted of 36 students of Grade 2013, while control group consisted of 30 students of Grade 2012. Precision medicine-based learning was applied in experimental group while traditional learning method was adopted by the control group. At the end of the course, students of two groups were implemented theoretical and experimental skills assessment; through questionnaire students were required to evaluate the effect of teaching methods and the number of two groups of students who asked questions after class greater than or equal to 1 times was counted to evaluate the students' learning enthusiasm. SPSS 15.0 software was used to make t test and Chi-square analysis for the data of the students. Results The assessment results showed that the experimental group was better than control group, especially in the section of comprehensive experimental design [(16.7 ± 2.04) vs. (13.9 ± 2.87), P=0.000]. The results from questionnaire showed that the satisfaction degree of experimental group was also higher than that of control group in many respects, including learning interests, innovation capability, knowledge mastery, cognition of precision medicine and clinical research, satisfaction with the teaching method (P<0.05). And students' learning enthusiasm and the proportion of the number of students asking questions in the experimental group were higher than the control group (P=0.000). Conclusions Precision medicine-based learning not only changes the situation of slavish imitation and passive acceptance in traditional learning, but also arouses students' interest in study and helps students to cultivate clinical thinking, which is com-mensurate to the characteristics of precision-medicine era.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

Expression and localization of adenovirus-mediated transcriptional factor Runx3 in malignant glioblastoma cells / 细胞与分子免疫学杂志

AIM: To construct the replicative defecient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells. METHODS: The runx3 gene with a flag tag was amplified by PCR using pCMV5-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMVRunx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn Ⅰ and Xho Ⅰ of pShuttle-CMV vector. This recombinant plasmid was linearized by Pmel and electronically transfected into BJ5183 cells to get the recombinant adenovirus vector Ad-Runx3. The recombinant adenovirus expressing Runx3 was infected into U251 malignant glioblastoma cells. The expression of exogenous Runx3 was observed by immonoblotting and its localization was detected by immunostaining using anti-Flag tag antibody. RESULTS: The recombinant adenovirus expressing Runx3 with a Flag tag was constructed and infected into U251 glioblastoma cells. The exogenous Runx3 protein was detected only in the nuclei. CONCLUSION: The recombinant adenovirus expressing Runx3 with a Flag tag is constructed successfully, and the Runx3 protein expressed in the nuclei of infected cells. The study laid a foundation for further research of the function of Runx3 in gliocarcinogenesis.

Construction and Expression of Recombinant Adenovirus Encoding Tumor Suppressor Fhit and Its Biological Function in Colon Cancer Cells / 中国生物工程杂志

AIM:To construct a recombinant adenovirus carrying Fhit gene,a tumor suppressor in many types of cancer,and to observe its biological function on the proliferation of colon cancer cells.METHODS:Fhit gene was cloned from the fetal liver cDNA library using the PCR method.The PCR product was inserted into the T vector to construct the plasmid pMD18T-Fhit.The Fhit fragment from the pMD18T-Fhit was inserted into the vector ptrack-CMV to construct a shuttle plasmid ptrack-CMV-Fhit.After PmeI digested and linearized process,ptrack-CMV-Fhit was co-transformed into Escherichia coli strain BJ5183 together with the adenovirus backbone vector pAdEasy-1 to generate a recombinant adenovirus plasmid by homologous recombination.The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein.Furthermore,the adenovirus rAd-Fhit was infected into colon cancer cells,and the expression of the ectogenic protein was detected by Western blotting.Finally,the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay.RESULTS:Constructed the recombinant adenovirus encoding Fhit gene and expressed it in colon cancer cells successfully.Detected that the proliferation of colon cancer cells was inhibited obviously in rAd-Fhit-infected cells with comparison to the control groups.CONCLUSION:Fhit may function as a tumor suppressor in colon cancer cells,and the adenovirus-mediated Fhit can be a novel strategy for the colon cancer therapeutics.

Knockdown of ClC-2 Gene Expression Inhibits the Growth of BT-325 Human Glioma Cells / 中国康复理论与实践

@#ObjectiveTo observe the growth of BT-325 human glioma cells after interfering volume-regulated chloride channel ClC-2 gene.MethodsTwo expression recombinant vectors of ClC-2 gene were designed and constructed. The primary plasmid, pSUPER.puro-shRNA, and the two recombinant plasmids, pSUPER.puro-shRNA-ClC-21 and pSUPER.puro-shRNA-ClC-22, were transfected into BT-325 cells by LipofectamineTM2000 (Groups: control, PP1 and PP2, respectively). The mRNA expression of ClC-2 gene was detected with reverse transcription polymerasse chain reaction (RT-PCR), the cellular survival was determined with MTT assay, and the cell cycle was measured with flow cytometry (FCM). ResultsClC-2 mRNA expression and the growth of the cells in PP1 and PP2 groups were significantly lower than that of control group. The cell cycle progression was blocked in G1 phase (PP1 and PP2 vs control,P<0.01). ConclusionThe growth of BT-325 human glioma cells is prevented by knockdown of ClC-2 gene expression, which may be one of the novel targets to inhibit growth of human malignant glioma cells.

Construction and expression of single chain variable Fv of anti-idiotype monoclonal antibody against Vibrio alginolyticus / 中国免疫学杂志

Objective:To clone the single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus.Methods:Total RNA was extracted from hybridoma cell 2F4 secreting MAb against vibrio alginolyticus and cDNA was amplified by retropolymerase chain reaction(RT-PCR),the expression vector pTAT-AL1 was constructed for the recombinant V_H-V_L expression.The transformed E.coli BL21 cells were propagated and induced by IPTG.Results:The V_H gene contained 369 base pairs and encoded 123 amine acid residues;The V_L gene contained 339 base pairs and encoded 113 amine acid residues;There were four FRs three CDRs and two characteristic cysteine residues in the V_H and V_L gene,respectively.ELISA results showed the ScFv retained almost the same antigen affinity and specificity as its parent monoclonal anitbody.Conclusion:The single chain variable Fv of anti-idiotype monoclonal antibody against vibrio alginolyticus was constructed successfully and expression products was found in the periplasmic space and inclusion bodies.This ScFv might be a new generation of gene engineering vaccine of the anti-idiotype monoclonal antibody against vibrio alginolyticus in fishery.

Strategies of tumor-targeted gene therapy / 中国癌症杂志

Targeting of tumor cells is crucial for gene therapy of malignant diseases.This can be achieved by tumor targeted gene transfer or tumor specific gene expression, as well as secretion of tumor targeted therapeutic molecules by autologous normal cells.Tumor targeted gene transfer is mediated by the recognition of a class of tumor specific antigens or receptors by corresponding vector fused antibodies or ligands, while therapeutic genes can be selectively expressed in tumor cells under the control of tumor or tissue specific promoters or enhancers, as well as the induction of certain physical, chemical or physiological factors.

Screening of phage antibodies against HBsAg from the constructed phage display library and sequencing of the positive clones / 中国病理生理杂志

AIM: Screening of phage antibodies against HBsAg from the constructed phage display library and sequencing of the positive clones.METHODS: RNAs were extracted from blood lymphocytes of a HBsAg-immunized volunteer. Then cDNAs were synthesized by RT-PCR. To construct the phage antibody display library, cDNAs of Fd fragments and ? chains of the antibodies were amplified through PCR and were inserted into vector pComb3H. Three rounds panning against coated HBsAg showed the specific enrichment of phage antibody. Positive clones were selected and sequenced.RESULTS: A phage antibody display library was constructed after amplifing of Fd fragments and ? chains of IgG1. Three Fab antibodies from the library were selected and sequenced. The result of sequencing showed that two of the three antibodies had the same Fd fragment and all of the three had the same light chain. CONCLUSION: The phage antibody display library was constructed successfully and three specific antibodies (Fab fragments) have been obtained from it.

DISTRIBUTION, CLONING AND AMPLIFICATION SEQUENCE ANALYSIS OF GnRH GENE IN CULTURED GASTRIC PARIETAL CELLS OF RATS / 解剖学报

Objective To study the distribution and sequence analysis of gonadotropin releasing hormone (GnRH) gene in cultured gastric parietal cells of rats. Methods The distribution of GnRH molecule and GnRH mRNA were observed out through immunohistochemical ABC methods and in situ hybridization methods in cultured gastric parietal cells of rats. After isolation of the total RNA from the parietal cells, RT PCR was conducted to obtain GnRH cDNA. Then, the products of PCR was purified, digested by the restriction enzyme of Hind Ⅲ and EcoR Ⅰ, and DNA fragments interests were cloned into pUC19 vector. The products of PCR were analyzed by sequenceing with Sanger's method after identified by PCR and digestion of restriction enzyme. Results Gastric parietal cells showed GnRH immunoreactivity, positive material was located in cytoplasm with negative nuclei. GnRH mRNA hybridized signals were also detected in cytoplasm with negative nuclei. The specific amplified band of GnRH mRNA was detected through agarose gel electrophoresis and gene sequence is identical to the GnRH which has been reported in rat hypothalamus.Conclusion Our data suggest that GnRH could be produced by gastric parietal cells of rats and may modulate physiological function of gastric parietal cells of rats.\;[