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Excellent doctor education training plan is an important policy applied by the gov-ernment to enhance the quality of medical personnel training in China. One of the important purposes is to improve the medical students' ability to apply and practice the medical professional English. In order to achieve this purpose, we have applied bilingual teaching for the students of excellent doctor in medical cell biology course. This article mainly introduces some reform measures , which involves training teacher, preparing textbooks and resources, establishing network database, exploring teaching modes, improving teaching methods and means, implementing diversified evaluation methods, and so on. At the same time, we have summarized some problems in the teaching process, and thus put for-ward improvement strategy.
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Background and purpose: High expression of telomerase and telomere stability are two common features in tumor cell. hTERT is a catalytic subunit of telomerase, TRF2 is extremely important to maintain the length and stability of telomerase. This study was to construct the recombinant adenovirus mediated shRNA to hTERT and TRF2, and to investigate the inhibitory effects of the vector by solo-inhibiting and connect-inhibiting in the MCF-7 cells, in order to present a new approach to the gene therapy for breast cancer. Methods: rAd-hTERT and rAd-TRF2 were constructed and the expression of hTERT mRNA and TRF2 mRNA were tested by FQ-PCR 48 hours after transfecting in MCF-7 cells. Apoptosis was observed by flow cytometry 1 to 6 days after transfection. Results: ①At 48 hours after transfection, the results of FQ-PCR showed that compared to PBS group, the expression of hTERT in rAd-hTERT group was obviously decreased and the inhibition ratio was about 86%, but TRF2 had not been obviously inhibited (P>0.05);the expression of TRF2 in rAd-TRF2 group was obviously decreased and the inhibition ratio was about 80%, but hTERT had not been obviously inhibited (P>0.05);in rAd-hTERT/rAd-TRF2 group, the inhibition ratio of hTERT and TRF2 were about 88% and 85%. Comparing rAd-hTERT/rAd-TRF2 group with rAd-hTERT group and rAd-TRF2 group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). Otherwise, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). ②The result of flow cytometry showed that apoptosis was induced at the first day after transfecting in rAd-hTERT group and rAd-TRF2 group, the most obvious apoptosis was in the 3rd to 5th days,at the peak in the 5th day, and decreased in the 6th day after transfection. The apoptosis ratio of rAd-hTERT group was 46.2%, rAd-TRF2 group was 43.5%. The apoptosis ratio of rAd-hTERT/rAd-TRF2 group was 46.2% at first day, 68.5% at the second day, the most obvious apoptosis was in the 3rd to 6th days and was 77.6% in the 6th days in rAd-hTERT/rAd-TRF2 group. There were significant differences in apoptosis ratio in solo-inhibiting and connect-inhibiting(P<0.05). In addition, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences in apoptosis ratio(P>0.05). Conclusion: ①Target sequence of RNAi which aimed at hTERT gene and TRF2 gene was designed efficiently, and the RNAi expression vectors were seen in vivo study efficiently and specifically inhibited the correspond gene expression and promoted cell apoptosis in MCF-7 cells. ②rAd-hTERT vector and rAd-TRF2 vector have no synergistical effect and antagoinstical effect on inhibiting hTERT gene and TRF2 gene mRNA expressing in MCF-7, but there was synergistical effect in terms of the induction of apoptosis. So association-RNAi-technique targeting to the genes of telomere length and stability can effectively promote tumor cell into apoptosis and inhibit breast cancer cell growth. RNAi technique of connecting correlation genes is a more effective gene therapy strategy.
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Objective To explore the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and human telomerase reverse transcriptase(hTERT) gene expression in MCF-7 cells,by the way of the construction of the RNA interference(RNAi) expression vector of hTERT gene.Methods An interference target sequence TGT TCA GCG TGC TCA ACT A,which aimed at hTERT gene,was designed and the small interfering RNA(siRNA) expression plasmid pGenesil-hTERT was constructed by means of recombination.The recombinant plasmid was transfected into the MCF-7 cells by LipofectamineTM 2000 after the identification of the recombinant expression vector pGenesil-hTERT in the light of electrophoresis after digestion and DNA sequencing,then RT-PCR analysis was used to detect the gene expression,and Western blotting analysis was used herewith to detect the protein expression of hTERT,and the growth of MCF-7 cells was tested by MTT assay.Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely,and the recombining plasmid was successfully constructed.RT-PCR and Western blotting analysis showed that hTERT gene expression and protein expression of pGenesil-hTERT group were obviously decreased in MCF-7 cells,and the growth of MCF-7 cells of pGenesil-hTERT group was also inhibited.Conclusion The endogenous hairpin siRNA produced by DNA plasmid is capable of mediating hTERT gene inhibition in MCF-7 cells effectively,and furthermore inhibiting the cells growth and decreasing the speed of cells proliferation.This research may act as the experiment evidence to the gene therapy of cancer.
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Objective To construct the RNAi expression vector of hTERT gene,and to investigate the inhibitory effects of the vector on the growth of mammary adenocarcinoma cell MCF-7 and the activity of telomerase.So as to present a new approach to the gene therapy for breast cancer which aim at inhibiting the telomerase activity.Methods An interference target sequence TGTTCAGCGTGCTCA ACTA which aimed at hTERT gene was designed and the siRNA expression plasmid pGenesil-hTERT was constructed by the means of recombination.Meanwhile a negative control recombinant-pGenesil-HK that did not aim at any gene was constructed by the same method.The recombinants expression vectors pGenesil-hTERT and pGenesil-HK were identified by electrophoresis after digestion and DNA sequencing.The two recombinants were transfected into the MCF-7 cells by lipofectamineTM 2000.The telomerase activity was tested by telomerase repeat sequence amplification and polyacrylamide gel electrophoresis.Flow cytometry was used to detect the cell apoptosis.Results The results of analysis and the sequencing identification confirmed that the target sequence was inserted into the predicted site precisely and the recombining plasmids were successfully constructed.The PAGE results showed that the mark traps of telomerase were obviously decreased and the telomerase activity of pGenesil-hTERT was obviously inhibited.The apoptotic rate of the pGenesil-hTERT group was significantly higher than that of the pGenesil-HK group(P