Objective To investigate the relationship between vascular injury of diabetic rats with hind limb ischemia and endothelial nitric oxide synthase(eNOS) uncoupling.Methods 15 SD rats were randomly divided into 3 groups:control group (n =5),diabetes group (n =5) and diabetes ischemia group (n =5).Two weeks after the diabetic rat model and hind limb ischemia of diabetic rat model were set up,Enzyme linked immunosorbent assay(ELISA) was used to detect nitric oxide(NO) expression in the rat femoral artery; Dihydroethidium(DHE) fluorescent probe was used to detect superoxide(O2-) production in the frozen section of femoral artery; and Western blot was used to examine GTP cyclohydrolase 1 (GTPCH-1)expression in femoral artery.Results ①Compared with the control group,NO expression of femoral artery in diabetes group was significantly reduced,diabetes ischemia group decreased more significantly,and the difference between the 3 groups had statistical significance(P =0.000,P =0.000,P =0.001).②Frozen section showed:compared with the control group,O2-expression of diabetes group significantly increased,but not as much as diabetes hind limb ischemia group.③Western blot results showed that compared with the control group,the expression of GTPCH-1 in diabetes group reduced,diabetes hind limb ischemia group decreased more significantly,and the difference between the 3 groups had statistical significance (P =0.001,P =0.000,P =0.012).Conclusion eNOS uncoupling is present in diabetic rats with hind limb ischemia and vascular injury.

Objective To explore the aging mechanisms of human adult adipose-derived stem cells (hADSCs) in vitro.Methods The hADSCs came from the production of normal patients with liposuction.The hADSCs were cultured in the 37℃ incubator and passed until the cells were senescent.The senescence-associated β-gal staining (SA-β-gal) and histone protein γH2AX staining were carried out in both young and old hADSCs.The quantitative real-time PCR (qRT-PCR) was conducted to check the difference of the transcription function of the young and old hADSCs.Results The cultured hADSCs would be the senescent cells when the PD number was more than 35.The aging cells became big and had large flat nuclei and much more lososomes in the cytoplasm.The SA-β-gal staining and γH2AX staining were mostly positive in the aging hADSCs (PD>35) compared with the self-renewing hADSCs (PD<20).The qRT-PCR results showed that the transcription levels of Nanog and Oct4 gene were very low in aging hADSCs compared with the self-renewing ones.Conclusions The hADSCs will be senescent (SEN) when cultured and passed in vitro for long time and the aging process is similar to that of stem cells in vivo.The aging model of hADSCs can be established by this method and will be very useful in the anti-aging drug, regeneration medicine, tissue engineering and stem cell therapy.

Background and purpose:It has confirmed that docetaxel, in combination with cisplatin or oxaliplatin have good effect in the treatment of advanced gastric cancer. This study in order to observe the clinical efficacy and adverse reaction of weekly docetaxel combined with cisplatin (DP) versus weekly docetaxel combined with oxaliplatin (DO) as the firrst-line treatment of advanced gastric cancer.Methods:A total number of 76 cases of advanced gastric cancer were randomly assigned 2 arms, 38 per arm. DP regimen group (docetaxel 35 mg/m2,ivgtt,dl,d8 combined with cisplatin 60 mg/m2, ivgtt,dl,repeated every 3 weeks) and DO regimen group(docetaxel 35 mg/m2,ivgtt,dl,d8 combined with oxaliplatin 120 mg/m2 ivgtt,dl,repeated every 3 weeks). Results:No significant difference was found between DP regimen group and DO regimen group on the objective RR(37%vs 41%),PFS (4.9vs 4.4 months) and OS (9.7vs 12.3 months). The main grade 3 or 4 toxicity in the DP and DO groups was neutropenia, DO was less associated with nausea and vomiting, but more associated with peripheral neuropathy than DP group. No signiifcant difference was found between DP regimen group and DO regimen group on the anemia, thrombocytopenia, diarrhea, alopecia (P>0.05).Conclusion:Weekly docetaxel combined with cisplatin (DP) shows similar efifcacy and toxicity compared with weekly docetaxel combined with oxaliplatin (DO) as the ifrst-line treatment of advanced gastric cancer and worthy of further study.

Objective To clone and characterize Profilin encoding genes in Amaranthus spinosus and to analyze the contribution of different amino acids in isoallergens to allergen antigenicity and tertiary structure. Methods The primers were designed according to the core sequences which were obtained by bioinformatic analysis of the known Profilin amino acid sequences, followed by gene cloning from the Ama- ranthus spinosus cDNA pool and subsequent confirmation by double-digestion, colony PCR and DNA sequen- cing. Antigenicity evaluation and tertiary structural modeling of the encoded protein were accomplished by online software MULTIPRED and SWISS-MODEL, respectively. Results Two panallergenic genes, named as PRF7 and PRF23, were acquired from Amaranthus spinosus. Sequence and structure analysis demonstra- ted that there was some discrepancy in tertiary structures of the encoded proteins, besides distinct difference in their amino acid sequences. PRF7 exhibited high homology with panallergen Profilins Q64LH0, with the identities 98%, whereas the homology of PRF23 and Q9XF42 (apple allergen) was 81%. Q64LH0 and PRF23 were modeled as 3nulA (Q42449) and lg5uB (Q9LE18), respectively. PRF23 exhibited distinct0 three dimensional structural difference in certain fragments compared with Q64LH0 and other Profilins. Though the average values of antigenicity displayed no difference between Q64LH0 and PRF23 on whole se- quences, the antigenicity of PRF23 on certain fragments was obviously lower than that of Q64LHO because of the alteration of some amino acids with different characters, implying the cause of lower incidence of hay fe- ver in South China than in North China. Conclusion Based on sequence analysis, antigenicity evaluation and tertiary structural modeling for Q64LH0 and PRF23, we obtained lots of useful information about the contribution of different amino acids to antigenicity and protein structures, thus would facilitate allergen ge- netic improvement by amino acid replacement.

Objective To investigate the application of the parietal branches of superficial temporal artery island flap in the complex scalp defects.Methods A parietal branches of superficial temporal artery island flap on the ectatic scalp flap was designed to repair the complex scalp defects in 25cases and the repairing effect was observed.Results The island flaps were survived completely in 24patients,in which 1 patient had partial necrosis because of the flap tension was too large,but healed after local dressing and debridement.After followed up 6～ 12 months,the color and texture of the flap were the same to the surrounding normal scalp,and the shape was satisfactory.The flap donor site of hair growth was good,with well healing and no obvious complications.Conclusions The parietal branches of superficial temporal artery island flap can repair the complex scalp defects with the flexible flap design and movement.The flap survives well and the repair area is large.The flap and the surrounding scalp connects good.Therefore,it is a good method strongly recommended for small area complex scalp defects repair in clinics.

Objective To investigate the mechanism of sleeve gastrectomy combined with modified jejunoileal bypass (SG/MJIB) in improving the blood glucose of Goto-kakizaki (GK) rats.Methods According to the random meter method,the 28 GK rats were divided into 4 groups:sleeve gastrectomy with modified jejunoileal bypass (SG/MJIB),sleeve gastrectomy (SG),modified jejunoileal bypass (MJIB) and sham operation (SHAM).The changes of weight,food intake,fasting blood glucose,oral glucose tolerance test (OGTT),insulin tolerance test (ITT),plasma insulin and gastrointestinal hormonal (ghrelin and GLP-1) were monitored before and 16 weeks after surgery respectively.Results After surgery,the fasting blood glucose level in SG/MJIB group was obviously lower than that in SG,MJIB and SHAM groups (P＜0.05).From the 2nd week after surgery,OGTT of the SG/MJIB group was obviously improved compared to SG,MJIB,and SHAM groups (P＜0.02).In our post-op erative study,the insulin levels in SG/MJIB group were lower than those in SG,and SHAM groups (P＜0.05).Compared to MJIB and SHAM groups,ghrelin levels of SG/MJIB group were significantly decreased (P＜0.001),while GLP-1 levels of SG/MJIB group were higher than those of SG and SHAM groups (P＜0.01).Conclusions SG/ MJIB can improve insulin sensitivity and insulin secretion,and this effect is independent of the body weight and food intake.This study further validates that the gastrointestinal hormones play an important role in the pathogenesis and treatment mechanisms of type 2 diabetes mellitus.

AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.

To study the mechanism of the innoxious touch-stimulus on the modulation of hyperalgesia and the expression of the C-fos and the nerve growth factor (NGF) receptor-TrkA in the spinal dorsal horn neurons following the chronic constriction injury (CCI) of the sciatic nerve in rats, 60female Sprague-Dawley rats were randomly divided into sham-operation group and CCI group, with each group being further divided into 3 subgroups on the 7th, 14 th and 28th day after operation (n=10). The mechanical and the thermal withdrawal threshold were assessed following the touch stiumulation after the CCI, immunohistochemical methods were employed to observe the expression of the C-fos and TrkA in spinal dorsal horn. Our results showed that the hyperalgesia appeared on the 4th day and reached the maximal level on the 14th day after operation. The expression of the C-fos also increased significantly and reached its maximal level on the 14th day after the touch-stimulus.Meanwhile, the TrkA expression was elevated significantly in both groups, as compared with basic data, and the difference was statistically significant (P＜0.05). It is concluded that the level of the C-fos expression changed with the paw withdrawal threshold variation and increased markedly following the innoxious touch-stimulus. The expression of the TrkA receptors also increased gradually following the development of the neuropathic pain. The results suggest that C-fos may play a crucial role in the development of the hyperalgesia in the earlier-time of the neuropathic pain, but TrkA receptors may be involved in the long-lasting adaptive changes of the central pathway in neuropathic pain.

Objective:To evaluate the diagnosis, surgical treatment, and neurological function recovery after surgery of patients with intraspinal tumors. Methods:The clinical data of 69 patients who suffered from intraspinal tumors and underwent surgery from Janu-ary 2008 to December 2012 were retrospectively analyzed. Neuroimaging and ASIA scoring were performed to examine the pathologi-cal characteristics of tumors and the neurological function of these patients before and after treatment. The major factors affecting prognosis were also probed, and the average follow-up period was 12.2 months. Results:Of the total cases, 62.3%showed intradural extramedullary intraspinal tumors located in the thoracic vertebra. Neurilemoma (Schwannoma) and meningioma were the most com-mon pathological types (53.5%). Posterior approaches with hemi-and complete-laminectomy were conducted to expose the intraspi-nal tumors, and the separation and removal of the tumors located at the cervical and thoracic levels were aided with surgical microsco-py. The main clinical symptoms, including back pain, radicular neuralgia, sensory disturbance, and motor dysfunction, were significant-ly improved after surgery, and this observation was supported by the follow-up ASIA scores before and after treatment. Of the in-volved cases, 91%were successfully treated, and their tumors were totally resected. Conclusion:Total or subtotal intraspinal tumor re-section enhanced with surgical microscopy could achieve satisfactory clinical results through posterior hemi-or complete-laminectomy.

OBJECTIVETo observe the impact of PDE5shRNA on cardiac remodeling and heart function following myocardial infarction in mice.

METHODSMyocardial infarction (MI) was induced in mice by left coronary artery ligation. Mice were randomly assigned to sham group (n = 6), PDE5shRNA group (n = 12), common adenovirus group (n = 15) and DMEM group (n = 8). Four weeks post-MI, the survival rate was evaluated. Cardiac function was examined by echocardiography. HE staining and Masson staining were used to evaluate the myocardial infarction size and fibrosis. The number of blood vessels was evaluated by immunohistochemistry, PDE5 protein expression in the left ventricular was detected using Western blot, level of cGMP or PKG activity in the left ventricle was evaluated with ELISA.

RESULTSFour weeks post-MI, all mice survived in the sham group, 3(37%) mice died in the DMEM group, 1 (8%) died in the PDE5shRNA group and 5 died in the common adenovirus group (33%). Infarct size was significantly reduced in PDE5shRNA group compared with the common adenovirus group and DMEM group [(25.4 ± 2.9)% vs. (42.0 ± 3.2)% and (43.4 ± 2.6) %, P < 0.05]. Cardiac function was significantly improved in PDE5shRNA group compared to common adenovirus group and DMEM group[LVFS: (21.1 ± 3.7)% vs. (14.2 ± 2.9)% and (14.22 ± 2.91)%, all P < 0.05; LVEF: (48.2 ± 7.1)% vs. (34.6 ± 6.2)% and (38.1 ± 2.8)%, all P < 0.05; LVESD: (3.87 ± 0.45) mm vs.(4.91 ± 0.62) mm and (4.63 ± 0.37) mm, all P < 0.05]. The blood vessel density was also higher in PDE5shRNA group compared with common adenovirus group (infarct area:14.3 ± 2.0 vs. 6.6 ± 1.2, P < 0.05; periinfarct area: 23.6 ± 2.1 vs. 13.7 ± 2.4, P < 0.05). Compared with common adenovirus group, level of PDE5 was significantly downregulated and level of cGMP or PKG was significantly upregulated in PDE5shRNA group (all P < 0.05).

CONCLUSIONSPresent study suggests PDE5shRNA improves cardiac function and attenuates cardiac remodeling through reducing infarction size and cardiac fibrosis and these beneficial effects are possibly mediated by activating cGMP/PKG signaling pathway.

Adenoviridae ; genetics ; Animals ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; genetics ; Disease Models, Animal ; Heart Failure ; etiology ; therapy ; Male ; Mice ; Mice, Inbred C57BL ; Myocardial Infarction ; complications ; RNA, Small Interfering ; genetics ; Ventricular Remodeling