Objective To construct the lentiviral vector to co-express enhanced green fluorescent protein (EGFP) gene and human insul in (insulin) gene, and to explore the condition to transfect human umbil ical cord mesenchymal stem cells (hUCMSCs) so as to lay a foundation for tissue engineered adipose reconstruction and transplantation in vivo infuture. Methods The insulin gene was cloned to lentiviral expression vector with EGFP [pLenti6.3-internal ribosome entrysite (IRES)-EGFP] by recombinant DNA technology, the positive clones were screened, and lentiviral packaged systems and target gene plasmid were co-transfected to package virus in 293T cells by lipofectin. The reporter gene expression was observed by fluorescent inverted phase contrast microscope, virus supernatant was collected, purificated and concentrated, and the titer of recombinant viruses was determinated. hUCMSCs from umbilical cord tissue of mature neonates were isolated and cultured by different multiple of infection (MOI, 0, 1, 3, 5, 7, 10, 15, and 20). By recombinant lentiviral infected hUCMSCs with reporter gene green fluorescent protein expression, the best MOI was screened; recombinant lentiviral infected hUCMSCs at the best MOI, then real-time PCR and Western blot methods were appl ied to detect insulin gene and insul in protein expression levels in cells. Results The recombinant lentiviral vector of co-expressing insulin gene and EGFP gene (pLenti6.3-insulin-IRESEGFP) was successfully constructed. Virus could be packaged, purificated and concentrated successfully. The virus titer was 1.3 × 108 TU/mL. The best MOI was 10 and the transfer efficiency was up to 90% in the same time. Real-time PCR results showed that insulin gene expression of transfected group was positive and non-transfected group was negative; Western blot detection confirmed that insul in protein expression of transfected group was positive in cells and supernatant, but that of non-transfected group was both negative. Conclusion Lentiviral vector pLenti6.3-insulin-IRES-EGFP carrying recombinant insulin gene could effectively transfect hUCMSCs and express insul in protein.
Objective To construct a recombinant adenovirus vector containing human vascular endothelial growth factor 165 (hVEGF165) [pAdxsi-enhanced green fluorescent protein (EGFP)-hVEGF165], and to observe the expression ofhVEGF165 by transfecting pAdxsi-EGFP-hVEGF165 into rat bone marrow mesenchymal stem cells (BMSCs) in vitro so as to lay a foundation for further research on gene therapy of blood vessel regeneration. Methods hVEGF165 was l iberated from plasmid and was subcloned into pShuttle-EGFP. The pShuttle-cytomegalo-virus-EGFP was then transferred to pAdxsi vector, by which pAdxsi-EGFP-hVEGF165 virus plasmid was obtained and was identified by enzymes restriction analysis and gene sequencing. The pAdxsi-EGFP-hVEGF165 was l inearized by digestion with restriction endonuclease PacI, and was then transfected into human embryonic kidney cells (HEK293). The retrieved recombinant adenovirus was titrated by using 50% tissue culture infective dose assay. The rat BMSCs were cultured and were infected with recombinant adenovirus containing EGFP (pAdxsi-EGFP). The multipl icities of infection (MOI) of transfection were determined by fluorescent inverted phase contrast microscope and flow cytometry (FCM), by which the most optimal value of MOI was confirmed and was used for transfecting pAdxsi-EGFP-hVEGF165 into BMSCs. The expression of hVEGF165 gene was indentified by performing Western blot, RT-PCR, and ELISA. The effect of transfection on BMSCs prol iferation was assessed by MTT. Results The expression of hVEGF165 cDNA in recombinant adenovirus plasmid was indentified by enzymes restriction analysis and gene sequencing. The titer of virus could be up to 1 ×1010 pfu/mL after several rounds of transfection and ampl ification. The efficiency of transfection on FCM was 88% when MOI being 150 pfu/ cell, at which the most optimal of MOI was achieved, as observed on fluorescence. The expressions of hVEGF165 at both mRNA and protein levels were detected after 48 hours of the transfection. The results of ELISA showed the expression ofhVEGF165 peaked at 7 days, and the production was found even after 20 days. Furthermore, the expression of hVEGF165 protein at 1, 3, 5, 7, 9, 11, 13, 15, and 20 days in the group transfected with pAdxsi-EGFP-hVEGF165 was significantly higher than that in the group transfected with pAdxsi-EGFP and in untransfected group (P lt; 0.05). The results of MTT demonstrated that here was no significant difference in absorbance (A) value between transfected with pAdxsi-EGFP-hVEGF165 group and untransfected group (P gt; 0.05). Conclusion BMSCs are suitable for gene transfection, and hVEGF165 gene can be transferred into BMSCs with high efficiency using pAdxsi-EGFP-hVEGF165 at a MOI of 150 pfu/cell. The transfected BMSCs can highly express hVEGF165, which has no effect on BMSCs growth and prol iferation.
目的 检测类风湿关节炎（RA）患者血清和关节液白细胞介素17A（IL-17A）的变化，探讨其与临床炎症指标、疾病活动性的关系。 方法 2011年6月－2012年6月采用酶联免疫吸附试验检测30例活动性RA患者和20例健康对照血清IL-17A水平，其中18例有膝关节积液RA患者同时检测配对血清和关节液IL-17A水平。 结果 RA组患者血清IL-17A水平显著高于健康对照组[（40.651 ± 16.402）、（23.799 ± 10.693） pg/mL，P＜0.05]。RA患者关节液IL-17A水平明显高于其血清中水平[（63.555 ± 23.405）、（43.727 ± 17.212） pg/mL，P＜0.05]。RA患者血清IL-17A水平只与疾病活动性评分（DAS28）呈正相关（r=0.498，P=0.020），而RA患者关节液IL-17A水平与DAS28和血清C反应蛋白有相关性（r=0.515，P=0.029；r=0.498，P=0.035）。 结论 RA患者血清和关节液IL-17A水平与疾病活动性显著相关，提示IL-17A可作为衡量疾病活动和关节损伤的标志之一。
ObjectiveTo analyze the effectiveness and problems existing in implementing acquired immune deficiency syndrome (AIDS) control and prevention in community health service centers, and to provide scientific evidence for promoting AIDS control and prevention. MethodsRelated information on AIDS control and prevention in community health service centers in a community in the whole year of 2013 was acquired. The effectiveness and problems existing in case management, laboratory testing and highly active antiretroviral therapy (HAART) based on the current community health service system were analyzed. ResultsBy the end of 2013, the rate of AIDS case management was from zero to 100%, the rate of CD4 T detection was from 76.60% (360/470) to 88.35% (508/575), and the rate of receiving highly active antiretroviral therapy among AIDS patients was from 81.40% (175/215) to 84.41% (287/340). But in the actual work on AIDS in community health service centers, there were still some common problems needing to be solved. ConclusionThe community level management mode of AIDS can promote the accuracy of AIDS patients' information, improve the rate of case management, the rate of CD4 T detection and HAART. It is suggested that the community level management mode of AIDS should be promoted in the cities where AIDS patients are concentrated.
ObjectiveTo investigate the efficacy of sulfasalazine combined with thalidomide in the treatment of spondyloarthropathy (SpA), and to probe whether the treatment can reach and maintain clinical remission for the patients. MethodBetween January 2011 and June 2013, we used a prospective, non-intervention and regular follow-up study to observe and assess 70 SpA patients on their Bath ankylosing spondylitis disease activity index, visual analogue scale score, peripheral arthritis, blood sedimentation, and C-reactive protein. All the patients had taken sulfasalazine, thalidomide and non-steroidal anti-inflammatory drugs for 24 weeks. Multivariate analysis of factors affecting the efficacy of the program was our object of this study. ResultsAfter 24 weeks, the total clinical remission rate of these patients was 72.9%. Clinical remission rate of 40 patients with short duration of SpA was 90.0%, while it was 50.0% for the other 30 patients with a non-short duration of SpA. Observation indexes before and after treatment in both groups of patients had significant differences (P<0.05). For patients with non-short duration SpA, the curative effect for female was obviously better than male, but the difference between male and female patients with short-duration SpA was not so obvious. Clinical remission rate for youth was similar with that for non-youth patients. Binary logistic regression analysis showed that whether the disease had a short duration[OR=3.408, 95%CI (1.637, 7.437), P=0.001] and whether the patients were urban residents[OR=4.163, 95%CI (2.011, 8.761), P=0.001] were statistically significant (P<0.05). ConclusionsClinical remission of spondyloarthropathy can be maintain by sulfasalazine combined with thalidomide. Clinical remission rate of the scheme is affected by duration of disease and residency except age and gender of the patients. Short duration and urban residency are independent factors for reaching clinical remission after treatment.
ObjectiveTo analyze retrospectively the risk factors of nonarteritic anterior ischemic optic neuropathy (NAION). MethodsThe complete clinical data of 116 patients (134 eyes) were collected. All patients were asked in detail about the disease history and symptoms and were examined for the visual acuity, intraocular pressure, fundus, visual field and fundus fluorescein angiography (FFA), blood pressure, blood glucose, blood fat and head MRI or CT. Suspicious cases and patients with incomplete clinical data were excluded. The relationship between NAION and age, visual field, FFA, systemic and ocular factors, onset seasons were retrospectively analyzed. Results80 patients (68.97%) were 55 to 70 years old. 97 patients (83.7%) had systemic diseases, including 38 patients (39.2) with diabetes mellitus, 32 patients (32.9%) with hypertension (8 patients had low blood pressure at night), 28 patients (28.9%) with hyperlipidemia, 16 patients (16.5%) with cerebrovascular diseases (mainly lacunar cerebral infarction), 6 patients (6.2%) with coronary heart disease. There were 8 patients with ocular factors, including 3 patients (2.6%) with cataract surgery history, 5 patients (4.2%) with small optic discs. The difference of percentage of with or without diabetes mellitus and hypertension was significant (χ2=362, 259; P < 0.05). There were 27.6% patients with disease onset at March to April, 24.1% patients with disease onset at September to October, much higher than other months (χ2=580, P < 0.05). Visual field test results showed that 49 eyes (36.5%) had inferior visual field defect, 12 eyes (9.0%) had superior visual field defect. FFA showed that in the early stage 103 eyes (76.9%) had optic weak fluorescence, 13 eyes (9.7%) had strong fluorescence; in the late stage, 110 eyes (82.1%) had strong fluorescence, 8 eyes (6.0%) had weak fluorescence. ConclusionsDiabetes mellitus, hypertension may be the system risk factors of NAION. The seasonal variation from spring to summer and from autumn to winter may also be another risk factor for the onset of NAION.
ObjectiveTo explore the new hospital management method about diagnosis-related groups (DRGs), and put forward some strategic suggestions.MethodsIn March 2019, using literature research method, relevant documents were consulted to understand the research policy and background. In April 2019, the DRGs data and first pages of medical records of a tertiary grade A hospital in 2018 were obtained through field survey. The DRG with the largest quantity of patients was selected, and then the top two treatment centers ranked by the quantity of patients were selected for analysis.ResultsA total of 11 936 patients’ face sheets for medical records were investigated, covering 18 major disease categories (MDCs) and 93 DRGs. Treatment center A and B were the top two treatment centers ranked by the quantity of patients, covering 8 MDCs and 34 DRGs. There were 1 116 patients in treatment center A and 470 patients in treatment center B, with the same case-mix index (0.820). There was no statistically significant difference in the average length of hospital stay between the two treatment centers (t=−1.926, P=0.054). The average hospitalization expenses [(45 902.64±30 028.22) vs. (40 763.34±25 141.12) yuan, t=−3.260, P=0.001], drug expenses [21 481.43 (10 663.16, 34 251.64) vs. 11 740.36 (5 818.37, 21 572.09) yuan, Z=−9.812, P<0.001], and other expenses [138.00 (84.00, 178.00) vs. 120.00 (72.00,155.28) yuan, Z=−3.573, P<0.001] in treatment center B were higher than those in treatment center A. But the medical technology expenses [(7 319.11±3 781.52) vs. (10 995.61±4 784.55) yuan, t=12.324, P<0.001] and nursing expenses [(578.42±226.82) vs. (882.99±781.63) yuan, t=8.187, P<0.001] in treatment center B were lower than those in treatment center A.ConclusionsThe disease diagnosis and treatment specifications need to be strengthened and the process needs to be optimized. In the next hospital management, we should pay attention to key indicators to improve performance appraisal, standardize the diagnosis and treatment process to promote clinical path, and mine deep data to make performance management detailed.
ObjectiveTo investigate the effectiveness of anterior cruciate ligament (ACL) reconstruction with the autologous ipsilateral peroneus longus tendon (PLT) under arthroscopy.MethodsA retrospective study was conducted on 35 patients with ACL rupture who underwent ACL reconstruction with autologous ipsilateral PLT under arthroscopy between October 2017 and October 2018. There were 19 males and 16 females with an average age of 43.4 years (range, 18-60 years), with 20 cases of left knee and 15 cases of right knee. The causes of injury included traffic accident in 14 cases, falling injury in 13 cases, and sports injury in 8 cases. The time from injury to operation ranged from 3 to 9 days (mean, 4.7 days). The patients suffered from swelling, pain, and limited mobility of knee joint before operation. The anterior drawer test, Lachman test, and pivot-shift test were positive before operation, whereas MRI was taken to confirm the ACL rupture. After operation, the patients were followed up every 3 months until the knee joint’s function returned to normal. MRI and X-ray films were used to observe the tendon-bone healing as well as the position of Endobutton suspension plate and hollow nail. The anterior drawer test, Lachman test, and pivot-shift test were conducted to observe the improvement of knee joint mobility. The functional improvements were evaluated by the International Knee Documents Committee (IKDC) score, Lysholm score, knee injury and osteoarthritis (KOOS) score.ResultsAll the 35 patients were followed up 12-18 months, with an average of 14.2 months. The incisions healed by first intention, and no complications such as infection, joint stiffness, and rerupture occurred. Postoperative anterior drawer test, Lachman test, and pivot-shift test turned to be negative of all patients. MRI showed that the ACL was continuous, and the tendon-bone in the distal femur tunnel and proximal tibia tunnel recovered well after operation. X-ray films showed that the positions of Endobutton suspension plate and hollow nail were stable. The IKDC, Lysholm, and KOOS scores at 3, 6, and 12 months after operation were significantly improved when compared with those before operation, and the scores were further improved with time after operation (P<0.05).ConclusionFor patients with ACL rupture, ACL reconstruction with the autologous ipsilateral PLT under arthroscopy has satisfactory effectiveness of quick recovery, good function, and great stability.