AIM: To determine the expression of c-kit mRNA and protein in the bladders in guinea pigs with diabetic cystopathy (DCP) and to explore the correlation and mechanisms between c-kit expression and DCP. METHODS: Sixty guinea pigs were divided randomly into control group (n=20) and experimental group (n=40). The guinea pigs in experimental group were injected with streptozotocin (STZ) to induce diabetes mellitus. After fed for 10 weeks, the animals in both groups were tested with urodynamics, and the guinea pigs in experimental group were divided into the subgroups of DCP and the diabetic no-cystopathy (NDCP) group according to the results of urodynamics. mRNA expression of c-kit was detected by reverse transcription polymerase chain reaction (RT-PCR) and protein expression of c-kit was tested and analyzed by laser scanning confocal microscope. RESULTS: Decreased expression of c-kit mRNA was observed in DCP group compared to control and the NDCP group. The ratio of c-kit mRNA and GAPDH was 5.66±0.54 in controls (P<0.05), 5.54±1.28 in NDCP group (P<0.05) and 4.65 ±0.47 in DCP group. c-kit protein expression significantly declined in DCP group. The mean value of fluorescence intensity was 856.52± 53.03 in control group, 844.67± 59.24 in NDCP group and 548.69± 48.51 in DCP (P<0.01).CONCLUSION: The declined expression of c-kit) gene at transcription and translation levels destroys the SCF/c-kit signal pathway, leading to the dysfunction of Cajal-like) cells in DCP guinea pig, so the abnormal expression of c-kit gene is involved in the pathogenesis of DCP.

Objective To discuss the types of ICC and characteristics of spontaneous Ca2+ waves of different types of ICC in the bladder of guinea pig. Methods Frozen-sections were made from the bladder of guinea pig and ICC were cultured in vitro. Cells were stained by indirect immunofluorescent method and detected by Laser scanning confocal microscope. The ICC cultured in vitro were divided randomly into 4 group: dimmer ICC,monomer ICC,dimmer ICC treated with 2-APB group and monomer ICC treated with 2-APB group according to the cell morphology and disrupted with 2-APB.The calcium concentration of ICC cultured in vitro were marked with Fluo-4 AM and disrupted by 2-aminoethoxydipheylbrate (2-APB, 100 μmol/L) in dimmer ICC treated with 2-APB group and monomer ICC treated with 2-APB group. The calcium oscillation function of ICC was observed under Laser scanning confocal microscope. Results For the monomer ICC and dimmer ICC in frozen sections and cultured in vitro,there were increased frequency (P＜0.01) and amplitude (P＜0.05) of spontaneous Ca2+ waves in dimmer ICC compared with the monomer monomer ICC. But after the cells disrupted by 2-APB after 15 min,There were decreased frequency (P＜0.01) and amplitude (P＜0.01) of spontaneous Ca2+ waves in the dimmer ICC treated with 2-ABP group compared with the dimmer ICC. The changes(P＞0.05) of spontaneous Ca2+ waves was not statistical significance in monomer ICC treated with 2-ABP group compared with monomer ICC group. Conclusions The bladder of guinea pig may exist 2 different types of ICC, dimmer ICC and monomer ICC. The excitability of spontaneous Ca2+ waves of dimmer ICC could be higher than in monomer ICC. The special structure of dimmer ICC may contribute to the formation of high spontaneous Ca2+ waves.

BACKGROUND: There is no study addressing transplantation of umbilical cord mesenchymal stem cells for the treatment of spinocerebellar ataxia.OBJECTIVE: To study the clinical effect of human umbilical cord stem cells transplantation in the treatment of autosomal dominant spinocerebellar ataxias. METHODS: Spinocerebellar ataxias patients selected from Stem Transplantation Center of the 455 Hospital of Chinese PLA were treated with umbilical cord mesenchymal stem cells transplantation via intrathecal injection. The number of umbilical cord mesenchymal stem cells was 107 per transplantation, once per week, for 4 weeks.RESULTS AND CONCLUSION: Both the total score of the International Cooperative Ataxia Rating Scale and Activities of Daily Living score were significantly decreased at 1 month after transplantation compared with before treatment (P < 0.05). The nerve function was significantly improved and the total effective rate was up to 16.7%. Experimental findings indicate that, transplantation of umbilical cord mesenchymal stem cells via intrathecal injection is a feasible and effective treatment to ameliorate the clinical efficacy of spinocerebellar ataxias patients and improve their quality of life.

To establish a detection method of oncolytic adenovirus/p53 and standard of quality control, human telomerase reverse transcriptase (hTERT) promoter, CMV fusion promoter containing hypoxia reaction element (HRE) and p53 gene were identified by vector DNA restriction enzyme digestion and PCR analysis. The result conformed that all modified regions were in consistent with theoretical ones. Particle number was 2.0 x 10(11) mL(-1) determined by UV (A260). Infectious titer was 5.0 x 10(10) IU mL(-1) analyzed by TCID50. In vitro p53 gene expression in human lung cancer cell H1299 was determined by ELISA, and A450 ratio of nucleoprotein in virus infection group to control group was 5.2. Antitumor potency was evaluated by cytotoxicity assay using human lung cancer cell A549, and the MOI(IC50) of this gene therapy preparation was 1.0. The tumor cells targeted replication ability of recombinant virus was determined by TCID50 titer ratio of filial generation virus between human lung cancer cell A549 and human diploid epidermal fibrolast BJ cells after infected by virus with same MOI. TCID50 titer ratio of tumor cell infection group to normal cell infection control group was 398. The IE-HPLC purity of virus was 99.5%. There was less than 1 copy of wild type adenovirus within 1 x 10(7) VP recombinant virus. Other quality control items were complied with corresponding requirements in the guidance for human somatic cell therapy and gene therapy and Chinese pharmacopeia volume III. The detection method of oncolytic adenovirus/p53 was successfully established for quality control standard. The study also provided reference for quality control of other oncolytic viral vector products.

Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.

Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes.Methods The differentiated mouse podocytes were exposed to contrast media (Iopromide,50 mg/L)、rapamycin (Rap,autophagy enhancer,1 ng/L),3-methyladenine (3-MA,autophagy inhibitor,2 mmol/L) for 2 hours.The expression of autophagy protein LC3-Ⅱ and Beclin-1 as well as oxidative stress-related proteins Catalase,MnSOD were detected by Western blot.The formations of autophagy were observed by MDC staining,and the levels of reactive oxygen species (ROS) by CM-H2DCFDA staining.Cell activity was evaluated by CCK8 assay.Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media,the expression of LC3-Ⅱ and Beclin-1 were enhanced,Catalase and MnSOD were inhibited (all P ＜ 0.05).Rapamycin increased the expression of Catalase,MnSOD and cell activity of podocytes,reduced the generation of ROS (all P ＜ 0.05),but in Rap group,cell activity showed no significant difference (P ＞ 0.05).3-MA decreased the expression of Catalase 、MnSOD and inhibited the cell activity of podocyte,increased the generation of ROS (all P ＜ 0.05).Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.

Objective: To evaluate the quality status of recombinant human interferon α2a injections and find out some quality problems. Methods:The statutory testing methods combining with the exploratory studies were used to examine the samples, and the quality status of recombinant human interferon α2a injections was evaluated by statistical analysis of the results. Results: All 28 bat-ches of the injections were qualified using the statutory testing methods. The exploratory studies showed that if the specific activity was determined, the qualified rate was only 87. 0%. All 7 batches of drug substances were qualified using the statutory testing methods. The exploratory studies showed that if the related protein was determined, the qualified rate was 57. 1%. Conclusion:At present the quality of recombinant human interferonα2a injections is generally good. The current standards are feasible;however, improvement is still needed. Specific activity determination should be supplemented the standards for drug products and related protein determination should be supplemented the standards of drug substances.

AIM: To study the tumor metastasis-related genes expression in adenomyosis and normal endometrium in order to investigate the pathogenesis of adenomyosis. METHODS: 43 specimens of adenomyosis, 22 specimens of controls (normal endometrium) were studied. The expressions of nm23-H1, MMP-2, MMP-9, MT1-MMP, and TIMP-1 in adenomyosis and controls were detected by immunohistochemical method. RESULTS: The expression levels of MMP-2, MMP-9, and MT1-MMP in adenomyosis were significantly higher than those in controls ( P 0 05). CONCLUSION: MMP-2, MMP-9, especially MT1-MMP, maybe play an important role in the pathogenesis of adenomyosis.

Objective To assess the effect of repetitive transcranial magnetic stimulation (rTMS) on motor and ambulatory function in incomplete spinal cord injury (SCI) patients. Methods 18 incomplete SCI patients (AIS D) were randomized to treatment group (n=10) and control group (n=8). The treatment group received rTMS while the control group received sham stimulation for 2 weeks. All the patients received routine rehabilitation. They were assessed with Lower Extremity Motor Score (LEMS), 10 m Walking Test for Walking Speed, modified Ashworth scale (MAS), Walking Index for SCI Scale II(WISCI II), and Spinal Cord Independence Measure (SCIM) before and after treatment, and followed up for 2 weeks after treatment. Results The treatment group significantly improved in LEMS, walking speed, and SCIM after treatment and during follow up (P<0.05), while the control group improved only in SCIM (P<0.05). There was more significant improvement in LEMS in the treatment group than in the control group (P<0.05) after treantment and during follow up. There was no difference between two groups in MAS, walking speed, WISCI II and SCIM. Conclusion rTMS can further improve the motor of lowere limbs for incomplete SCI patients.

Objective: To investigate the relationship between neutrophil to lymphocyte ratio (NLR) and estimated glomerular filtration rate (eGFR) in patients with type 2 diabetes mellitus. Methods: The clinical data of 117 patients with type 2 diabetes mellitus from January 2016 to June 2017 in Anhui No.2 Provincial People′s Hospital were analyzed retrospectively. According to the eGFR level, the patients were divided into 3 groups: eGFR ≥ 90 ml/(min·1.73 m²) in 68 cases (DM0 group), eGFR 60 to 89 ml/(min·1.73 m²) in 33 cases (DM1 group), and eGFR<60 ml/(min·1.73 m²) in 16 cases (DM2 group). In addition, 30 healthy people in the same period were selected as control group (NC group), eGFR ≥ 90 ml/(min·1.73 m²). The systolic blood pressure, diastolic blood pressure, blood routine, glycosylated hemoglobin (HbA₁c), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), urea nitrogen, creatinine and uric acid were recorded; and the NLR was calculated. The influencing factors of eGFR in patients with type 2 diabetic mellitus were analyzed, and the relationship between NLR and eGFR was evaluated. Results: Compared with that in NC group and DM0 group, the eGFR in DM1 group and DM2 group was significantly lower: (75.12 ± 8.14) and (46.31 ± 13.25) ml/(min·1.73 m²) vs. (114.17 ± 12.21) and (113.21 ± 12.04) ml/(min·1.73 m²), the NLR was significantly higher: 2.50 ± 1.16 and 2.75 ± 1.39 vs. 1.53 ± 0.22 and 1.83 ± 0.65, and there were statistical differences (P<0.05). Compared with that in DM1 group, the eGFR in DM2 group was significantly lower, the NLR was significantly higher, and there were statistical differences (P<0.05). There were no statistical differences in NLR and eGFR between DM0 group and NC group (P>0.05). Correlation analysis result showed that NLR, age, course of disease, systolic blood pressure, TC, HDL-C, urea nitrogen, creatinine and uric acid were negatively correlated with eGFR (r=-0.415, -0.555, -0.491, -0.432, -0.259, -0.237, -0.584, -0.840 and -0.261; P < 0.05); gender, diastolic pressure, HbA₁c, TG and LDL-C were not correlated with eGFR (P > 0.05). Multiple linear regression analysis result showed that NLR, age, TC, creatinine and systolic blood pressure were independent risk factors of eGFR in patients with type 2 diabetes mellitus (P<0.01 or < 0.05). Conclusions There is a close relationship between the increase of NLR and the decrease of eGFR in patients with type 2 diabetes mellitus. Monitoring NLR is helpful to understand the changes of eGFR in patients with type 2 diabetes mellitus.