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

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.

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.

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.

Objective To analyze the clinical features of patients with sporadic adult-onset neuronal intranuclear inclusion disease (NIID),and raise awareness of the disease among medical workers.Methods The clinical data of two patients with pathologically confirmed adult sporadic NIID,admitted to our hospital in February 2018 and October 2018,were collected.The clinical manifestations,head MR imaging,cutaneous pathological features,treatments and prognoses were retrospectively analyzed.Results Both patients were characterized with slow progressive dementia,accompanied with diverse clinical manifestations involving the central and autonomic nervous systems.Acute encephalopathic signs occurred in both patients.Head MR imaging showed extensive leukoencephalopathy mainly in the frontal and parietal lobes;these white matter abnormalities showed hyperintensity in T2-weighted imaging and liquid attenuated inversion recovery sequences,and equal or low signal in T1-weighted imaging.Remarkably,specific curve-like high-intensity signals along the corticomedullary junction in the bilateral frontal lobe were both observed in diffusion-weighted imaging.Patient 2 with seizures showed unilateral cerebral cortical edema on head MR imaging.In both patients,skin biopsy revealed specific eosinophilic inclusion bodies in the nucleus of some sweat gland cells,adipocytes and fibroblasts.Patient one was treated with dexamethasone intravenous drip to relieve headache and vision loss,and cognitive therapy was given.The acute encephalopathy of patient two was relieved by intravenous gamma globulin.Conclusions NIID has various clinical manifestations of central,peripheral,and autonomic nerve systems.Head MR imaging characteristic changes and skin pathological biopsy contribute to the diagnosis.Immunomodulatory therapy may be effective for acute encephalopathic symptoms in NIID.

Objective:To evaluate the clinical efficacy of endoscopy for early cancer of duodenal papilla.Methods:A retrospective analysis was performed on data collected from 23 consecutive patients with early cancer of duodenal papilla, who underwent endoscopic treatment from January 2015 to January 2021 in Beijing Friendship Hospital. Baseline data, endoscopic and pathological data, occurrence and outcome of complications were studied.Results:Twenty-three patients successfully received endoscopic treatment. The maximal diameter of lesions evaluated under endoscopy was 1.90±0.83 cm. Among the 23 cases, 20 underwent endoscopic mucosal resection and 3 underwent endoscopic piecemeal mucosal resection. Delayed bleeding occurred in 5 cases (21.7%), 3 patients (13.0%) developed postoperative hyperamylasemia, 6 patients (26.1%) developed mild acute pancreatitis, and 1 patient (4.3%) had pancreatic duct stent displacement after the operation, which improved after medical or endoscopic treatment. No perforation occurred during the perioperative period. In terms of final pathology, the en bloc resection rate was 82.6% (19/23), and the complete resection rate was 78.3% (18/23). Preoperative endoscopic ultrasonography showed that 19 lesions were confined to the mucosal layer, which were all demonstrated by postoperative pathology. Four other cases were suspected to be involved in the submucosa or the end of the pancreaticobiliary duct under endoscopic ultrasonography, two of which were confined to the mucosal layer, and the other 2 cases involved the submucosal layer, so additional surgery was performed. A total of 18 patients were followed up, among whom 14 achieved complete resection of postoperative pathology, and 2 patients (14.3%, 2/14) were found to have recurrence at 12 and 51 months respectively after the treatment and did not relapse after surgical treatment and endoscopic treatment respectively. Among 4 other patients of follow-up whose pathology did not achieve complete resection, 1 had no recurrence, and the other 3 received additional surgical treatment without recurrence.Conclusion:Endoscopic treatment for early cancer of duodenal papilla is safe and effective. It is necessary to improve preoperative evaluation, stay alert to perioperative complications, and pay attention to regular postoperative endoscopic follow-up.