BACKGROUND:Human mesenchymal stem cells (hMSCs) become aging and even die after several passages. Some investigations have shown that telomere has a close correlation with life span of the cells. Whether the ectopic expression of human telomerase reverse transcriptase (hTERT) could induce the activity of the telomerase, maintain the length of telomere, and finally prolong the life cycle of MSCs without losing their multipotent differentiation capacity is still uncertain.OBJECTIVE: To observe the influence of the ectopic expression of hTERT on the telomerase activity and cell life cycles of hMSCs.DESIGN: Repetitive measurement trails.SETTING: Research Center of Stem Cell, Zhengzhou University Medical College.MATERIALS: The experiment was conducted in the Research Center of Stem Cell, Zhengzhou University Medical College from October 2003 to December 2005. hMSCs were obtained from 20 healthy donators from the Department of Pediatric Surgery and Outpatient, the Third and First Affiliated Hospitals of Zhengzhou University. Enhanced green fluorescent protein plasmid (pEGFP-C1) and pEGFP-hTERT were provided by Dr. Chantal Autexier of Canada. DH5α strain provided by Dr. Hou Wei-hong, the Key Molecular Medical Laboratory of Zhengzhou University Medical College.METHODS.: Under sterile condition, 2 mL bone marrow of sternum of healthy donors were harvested, and prepared after centrifugalization,dilution and passage.① Transfection of pEGFP-hTERT into hMSCs and the screening and amplification of resistance cloning:The 5th passage cells were seeded in a 24-well plate,and transfected by pEGFP-hTERT with lipofectamine method.The cells were divided into four groups including untransfected group,lipofectamine group,pEGFP-C1 group and pEGFP-hTERT group. Resistance cloning screen and amplification was performed by G418. ②hTERT mRNA expression and detection of telomerase activity:RT-PCR and PCR-ELISA were used to detect the hTERT mRNA expressions of the fifth passage hMSCs transfected with pEGFP-hTERT, and pEGFP-C1, the untransfected tenth passage hMSCs and K562 cells (positive control), and the telomerase activity of the fifth and thirtieth passage hMSCs transfected with pEGFP-hTERT,the fifth pEGFP-C1-transfected cells and the tenth passage untransfected cells. ③Karyotype analysis of hTERT-transfected MSCs: Chromosome analysis was performed by conventional Giemsa staining.④Inducement of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification:The transfected MSCs were cultured in a medium containing epidermal growth factor and basic fibroblast growth factor, which could induce the cells differentiate into neuron-like cells. The culture solution was changed every 3 days, and the changes in cell growth condition and morphologic characteristics were observed under an inverted microscope. The microtubule associate protein (MAP2) and neurofliament subunit M (NF-M) were identified by RT-PCR.MAIN OUTCOME MEASURES:①hMSCs transfection with different kathion liposomes and the screening and amplification of resistance cloning; ②hTERT mRNA expressions of each group and detection of telomerase activity; ③Karyotype analysis of pEGFP-hTERT-transfected MSCs; ④ Induction of differentiation from telomerase-positive MSCs into neuron-like cells and RE-PCR identification.RESULTS: ①With the decrease of G418 concentration, the cells in the untransfected and lipofectamine groups died, and stably EGFP expressed MSCs were obtained; after G418 screening, there was a cell clone undergone 35 passages and continued to proliferate, whose appearance and growth characteristics were similar to the untransfected MSCs observed under inverted microscope. ②The fifth passage pEGFP-C1-transfected hMSCs and tenth passage untransfected hMSCs remained telomerase-negative, but the K562 and fifth passage hTERT-transfected cells showed positive telomerase activity. ③The telomerase activity of the fifth and thirtieth passage hTERT-transfected cells was positive. ④The hTERT-MSCs at passage 10, 20 and 30 had 23 pairs of chromosomes, and two X chromosomes. So they were still normal diploid with normal chromosome appearance and number. ⑤Many hTERT-transfected MSCs had the typical appearance of neuron-like cells. RT-PCR analysis showed that th expressions of MAP2 and NF-M were increased.CONCLUSION:Ectopic expression of the hTERT gene is found in hMSCs,and can induce the telomerase activity of hMSCs.The ectopic expression of the hTERT gene in hMSCs could extend the life spans of cells and maintain their multipotent differentiation capacity.

BACKGROUND: Stem cells are relatively primitive cells possessing the capabilities of self-renewal, high proliferation and multi-potential differentiation in vivo under certain conditions. Pancreatic stem cells and umbilical cord blood mesenchymal stem cells (MSCs) may serve therapeutic purpose clinically, but they are still difficult to culture in vitro at present.OBJECTIVE: To explore the method for isolation, purification and culture of pancreatic stem cells and umbilical cord blood MSCs in vitro and observe their morphological changes during culture in vitro.DESIGN: Completely randomized experiment with repeated measurement.SETTING: Stem Cell Research Center, Teaching and Research Division of Physiology, Medical School of Zhengzhou University.MATERIALS: This experiment was conducted in the Stem Cell Research Center, Teaching and Research Division of Physiology, Medical College of Zhengzhou University, between April 2004 and January 2005. Ten to fifteen newborn SD rats (1-3 days) were selected for culture in vitro of pancreatic stem cells, and fresh umbilical cord blood was collected from healthy woman (24-35 years old, with informed consent) at full-term delivery for culture in vitro of umbilical blood SMCs.METHODS: The abdomen of the newborn SD rat was opened under aseptic condition to obtain the pancreas, which was cut into small tissue blocks and digested with type-V collagenase for islet isolation. The isolated islets were purified in continuous roller-bottle culture. Umbilical cord blood was freshly collected for isolating the monocytes by means of density gradient centrifugation in lymphocyte separation medium (with density of 1.077 g/cm3). The islet cells and umbilical cord blood monocytes were cultured in the incubator at 37 ℃ with 5% CO2. The morphological changes of the cells were observed at designed time points and flow cytometry was used to determine the expression of cell surface molecules.MAIN OUTCOME MEASURES: The isolation and culture of pancreatic stem cells and umbilical cord blood MSCs, and their morphological changes during culture in vitro.RESULTS: During culture in vitro, the fusiform islet progenitor cells showed adherent polar growth and continuous proliferation, which covered the whole bottom of the flask after 12-14 days and could be subcultured for passages. However round cells appeared after removal of the growth factor and serum in the culture medium. The monocytes isolated from the umbilical cord blood grew initially into numerous hematopoietic cell clones, most of which proved to be granulocyte clones by Switzerland staining. Seven days later, flat flask wall-adhering epithelial cells and long fusiform fibroblasts were observed mixed with a number of osteoclasts. As the cell culture was prolonged, the cell number increased steadily.CONCLUSION: Pancreatic stem cells and umbilical cord blood SMCs can be cultured in vitro for further experiments.

Objective To establish the biological reference interval of procalcitonin(PCT) applicable for children in Chongqing.Methods Serum PCT level were detected in 120 healthy children with age from 0 to 16 years, including 73 cases of male and 47cases of female children, by using by Maglumi2000 plus PCT analysis system.All data was evaluated according to EP28-A3c document to establish the biological reference interval.Results Data of PCT levels were with non-normal distribution, and without statistical difference between children of different ages and genders(P>0.05).The biological reference interval of PCT was less than or equal to 0.038 μg/L.Conclusion It might be important to establish a usefully biological reference interval in different laboratories with relative detection system, especially for children.

Objective To investigate the relationship between corneal basal nerve change and type 2 diabetic retinopathy based on confocal laser microscopy.Methods Together 118 patients with type 2 diabetes (T2D) were collected in our hospital from February 2016 to February 2017,including 57 patients with diabetic retinopathy (DR group) and 61 patients without DR (NDR group).For comparison,60 healthy volunteers were selected as the control group.And all the subjects were examined by corneal confocal laser microscopy to analyze the relationship between the morphological parameters of the corneal nerve and clinical variables.Results Corneal nerve fiber density,corneal nerve branch density and corneal nerve branch length in DR group were (20.03 ±4.22) · mm-2,(22.01 ± 7.05) · mm-2 and (9.50 ± 1.76) mm ·mm-2,significantly less than those of the control group and NDR group (all P ＜ 0.05);and corneal nerve fiber curvature was (0.30 ± 0.03),significantly higher than that of the control group and NDR group (all P ＜ 0.05);In DR patients,phase Ⅲ patients had smaller the corneal nerve fiber density,corneal nerve branch density and corneal nerve branch length,but the larger corneal nerve fiber curvature than the phase Ⅰ and Ⅱ patients (all P ＜ 0.05);course of disease of DR group was (12.04 ± 2.48) years,which was significantly higher than that of NDR group (P ＜ 0.05),while fasting C peptide and fasting insulin were (1.41 ± 0.58) μg · L-1 and (20.05 ± 7.91) mU · L-1,respectively,significantly lower than those of NDR group (all P ＜ 0.05);The duration of T2D was negatively correlated with the corneal nerve branch density and corneal nerve branch length (r =-0.322,-0.317,all P ＜0.05);Fasting C peptide was positively correlated with the corneal nerve branch density (r =0.298,P ＜ 0.05),and negatively correlated with the corneal nerve curvature (r =-0.311,P ＜ 0.05).Conclusion Patients with T2D retinopathy have abnormal morphology of corneal nerve.And confocal laser scanning microscopy is conducive to the early detection of microvascular disease in T2D patients with a longer course of disease or a low level of fasting C peptide.

Based on the theory of the rise and fall of qi-blood in meridians, Professor GAO Yu-chun harmonizes the qi-blood and yin-yang for diagnosis and treatment of hypertersion from liver, spleen and kidney. With reference of the academic idea of“Preventive Acupuncture”, he investigated the qi and blood, treated diseases in the upper part by managing the lower part, pay attention to the syndrome differentiation of excess and deficiency, the different of acupoint property, and the treatment of acupuncture or moxibustion. Thus, through balancing yin and yang, the qi and blood are regulated to reach the therapeutic aim of“yin and yang in equilibrium”.

Objective To investigate the mechanisms and clinical characteristics of fatal asthma attack induced by amiodarone in the elderly,and to discuss its serious potent risks.Methods We reported two cases of fatal asthma attack in the elderly with fast atrial fibrillation induced by amiodarone,and discussed the clinical manifestation and emergency treatment,with literatures review.Results The two patients presented with respiratory arrest.After targeted therapies,including artificial ventilation and hormone injection,the patients were capable of spontaneously breathing,while still in severe asthma attack.And further anti-asthma treatment led to a complete remission.Conclusions Amiodarone may cause extremely severe asthma attack in elder patients with tachyarrhythmia complicated with bronchial hyper-responsiveness diseases such as asthma or chronic obstructive pulmonary diseases.Clinicians should pay great attention to this situation and be prepared to cope with serious adverse reactions.

Child ; Cochlear Implantation ; Cochlear Implants ; Evoked Potentials, Auditory, Brain Stem ; Hearing Loss, Central ; physiopathology ; Humans ; Vestibulocochlear Nerve Diseases ; physiopathology

AIM: To construct a recombinant retrovirus vector carrying hTERT for establishing UCBMSCs with hTERT(hTERT-MSCs) to overcome their limited life span and detecting whether telomerized UCBMSCs line maintained long-term self-renewal and differentiation capacity.METHODS: The whole cDNA was generated by PCR amplifications from the plasmid pEGFP-hTERT-C1.The hTERT segments were subcloned into pLNCX2.The target cells were infected with these retroviral particles.The stably transfected cells were selected by neomycin and expanded life span which were designated hTERT-MSCs was observed.The expression of hTERT in mRNA level was detected by RT-PCR and the telomerase activity was measured by TRAP(PCR)-ELISA assay.The hTERT-MSCs were induced with 5-azacytidine to cardiac muscle cells and the specific marker of myocardiocyte was detected.RESULTS: The constructed plasmids were digested with restriction endonucleases(BglⅡand NotⅠ).Two characteristic segments including 6.1 kb and 3.6 kb were obtained.The hTERT-MSCs expressed hTERT in mRNA level.The telomerase activity of hTERT-MSCs was positive.The growth kinetics of hTERT-MSCs was higher than those in UCBMSCs.The hTERT-MSCs were induced to myocardiocyte.CONCLUSION: The hTERT recombinant retrovirus vector has been successfully constructed.The hTERT gene activates the telomerase and prolongs the life-span of cells.No effect of hTERT gene on some type of differentiation potential of MSCs is present.

The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.

Objective:To analyze the EGFR gene polymorphism in the non-small cell lung (NSCLCs) cancer in southern of Shaanxi Province.Methods:The next generation sequencing technology was used to detect the mutation of exon 18,19,20 and 21 of EGFR gene.We analyzed EGFR gene mutation rate in NSCLCs patients.233 patients was involved in our study.Results:82 cases with EGFR gene mutations was found,the mutation rate of exon18,19,20 and 21 was 1.3％,16.3％,0％ and 18％ respectively.The mutation rate of EGFR in male patients was lower (31.2％,39/125) than that of female cases (39.8％,43/108),the mutation rate of squamous cell carcinoma (22％,9/41) was lower than that of adenocarcinoma (39.1％,75/192).Conclusions:NSCLCs patients from southern Shaanxi Province had high mutation rate ofEGFR gene,and exon 19 and exon 21 mutations were in the majority.EGFR gene mutation rate was not related to gender and pathological types.