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.
目的：機械分離、培養小鼠耳蝸螺旋神經元，并進行免疫熒光細胞學鑒定，為后期進一步的實驗研究提供實驗材料。方法：采用初出生1～5天以內的昆明小鼠進行解剖、機械分離以獲得螺旋神經節組織，進行原代培養后，應用神經微絲蛋白（Neurofilament protein,NFP－H）單克隆抗體進行免疫熒光細胞學鑒定。結果：機械分離后獲得的螺旋神經節組織中的螺旋神經元，在體外培養條件下可以存活并進行正常分化。典型的螺旋神經元，其細胞形態呈橢圓形,胞體透明光滑、接近生理形態。熒光染色標記后，胞體和神經突起均顯色好，Schwann細胞和成纖維細胞未著色。結論：應用機械分離的方法獲得小鼠耳蝸螺旋神經節組織并進行培養，耳蝸螺旋神經元在體外可以穩定地存活生長。培養獲得的細胞形態和生存狀態接近生理狀態，滿足電生理、免疫細胞化學、藥理學等研究。應用特異性的神經微絲蛋白對培養獲得的螺旋神經元進行免疫熒光細胞學鑒定，特異性好，熒光顯色好。
Objective To investigate the impacts of cytokines （interleukin-4，IL-4；tumor necrosis factor-α，TNF-α） and medications of bronchial asthma （dexamethasone，aminophylline，salbutamol） on the activity of histamine N-methyltransferase（HMT） in tracheal epithelial cells.Methods BEAS-2B bronchial epithelial cells were cultured and treated with different concentration of TNF-α, IL-4, dexamethasone, salbutamol and aminophylline respectively. The activity of HMT in BEAS-2B cells was determined by high performance liquid chromatography.Results The activity of HMT in tracheal epithelial cells was (50±7) pmol?min-1?mg pro-1.TNF-α and IL-4 lowered the activity of HMT significantly at the concentration equal to or higher than 1 ng/mL and 5 ng/mL respectively，and reached the maximum inhibitory effect at the level of 10 ng/mL.Dexamethasone and aminophylline could ameliorate distinctly the inhibitory effect of TNF-α on the activity of HMT, while salbutamol had no significant inhibitory effect.Conclusions TNF-α and IL-4 exert the lowering effect on the activity of HMT，which would be one important cause of airway hyperreactivity.Glucocorticoids and theophyllines are administered to treat asthma partly due to its relieving mechanism of TNF-α negative effects on HMT.
ObjectiveTo explore the effect of fingolimod (FTY720) on secondary nerve injury after thalamic-ventricle hemorrhage (TH-IVH) in rats.MethodsAdult male Sprague Dawley rats (clean animal) were randomly divided into 3 groups: sham group, TH-IVH group, and intervention group (FTY720 group), with 6 rats in each group. TH-IVH model was established in both TH-IVH group and FTY720 group, but only the rats in FTY720 group were treated with FTY720. The observation was conducted at the 1st, 3rd and 7th day after modeling. The main observation index included scores of neurological function, change of body weight, water content of brain tissue, the activation of inflammatory cells, the degree of neuronal degeneration and apoptosis, and the level of cell autophagy.ResultsAt the 1st, 3rd and 7th day after modeling, the change of body weight, the neurological score, brain edema and microglia activation in TH-IVH group were statistically different from those in sham group and FTY720 group (P<0.05). The number of degenerated neurons and the number of apoptotic cells in TH-IVH group were statistically different from those in sham group and FTY720 group at the 1st and 3rd day after modeling (P<0.05). The differences in the ratio of LC3Ⅱ/LC3Ⅰ protein expression andBcl-2/Bax expression were statistically significant between FTY720 group and TH-IVH group at the 1st and 3rd day after modeling (P<0.05).ConclusionsFTY720 can improve neurological function of the TH-IVH model in the acute phase, and has certain neuroprotective effect. The neuroprotective effect of FTY720 may be associated with neuronal autophagy and apoptosis regulation and immunosuppression.
Objective To explore whether bundled care for anesthesia management can reduce the risk of postoperative nausea and vomiting (PONV). Methods The data of laparoscopic cholecystectomy patients admitted to the Day Surgery Center of West China Hospital, Sichuan University between July and November 2021 were retrospectively collected. Patients were divided into a bundled care group and a control group based on whether anesthesia management was implemented according to the bundled care. The demographic characteristics, intraoperative anesthesia management methods, postoperative conditions, and incidence of PONV between the two groups of patients were analyzed and compared. Results A total of 314 patients were included. Among them, there were 124 cases in the bundled care group and 190 cases in the control group; PONV occurred in 52 cases, the incidence of PONV was 16.6% (52/314). Except for surgical time and postoperative incision infiltration (P>0.05), there were statistically significant differences in age, gender, body mass index, anesthesia time, airway establishment, and postoperative analgesic use between the two groups of patients (P<0.05). There was no statistically significant difference in the occurrence of PONV between the bundled care group and the control group (17 vs. 35 cases; χ2=1.205, P>0.05). The results of logistic regression analysis showed that PONV was correlated with gender [odds ratio=0.107, 95% confidence interval (0.030, 0.375), P<0.001], and using bundled care [odds ratio=0.388, 95% confidence interval (0.169, 0.894), P=0.026]. Conclusions Women are at high risk of PONV among patients undergoing day laparoscopic cholecystectomy. The risk of PONV is lower when using bundled care.
目的 回顧性研究老年系統性紅斑狼瘡（SLE）患者合并感染的危險因素。 方法 選取1995年1月－2009年12月間在四川大學華西醫院確診為SLE，起病年齡為50周歲以上的158例患者，收集性別、臨床表現、疾病活動度、實驗室檢查指標、合并癥以及并發癥等進行單因素分析或多因素非條件logistic回歸分析。 結果 所納入的158例患者中，合并感染53例（占33.5%），采用單因素分析顯示疾病活動性（P=0.001）、低蛋白血癥（P=0.030）、糖尿病（P=0.003）、肺間質病變（P=0.000）與老年SLE患者感染發生有關。經logistic回歸分析顯示，疾病活動性（OR=7.533，P=0.000）、肺間質病變（OR=19.762，P=0.000）、糖尿病（OR=6.862，P=0.025）是老年SLE患者感染發生的危險因素。 結論 積極控制老年SLE的疾病活動度，減少危險因素的發生是控制老年SLE患者并發感染的有效手段。
Objective To study the influence of different mechanical environments on repair cartilage defect with marrow mesenchymal stem cells as seed cells. Methods The rabbit marrow mesenchymal stem cells were isolated and cultured. The cartilage defects were repaired by autologous tissue engineered cartilage with the marrow mesenchymal stem cells as seed cells. Fifteen rabbits with cartilage defect were divided into 3 groups: dislocation group with cell-free scaffold(controlgroup), dislocation group with cartilaginous construct and normal mechanical environment group with cartilaginous construct. The repaired tissue was harvested and examined 6 weeks postoperatively. Results The repair tissue in normal mechanical environment group with cartilaginous construct showed cartilage-like tissue in superficial layer and subchondral bone tissue in deep layer 6 weeks postoperatively. The defect was filled with bone tissue in dislocation group with cartilaginous construct 6 weeks postoperatively. The surrounding normal cartilage tissue showed vascular invasion from subchondral area and the concomitant thinningof the normal cartilage layer. The cartilaginous construct left in the femoral trochlea groove formed hyaline cartilage-like tissue. The defect was repaired byfibrous tissue in control group. Conclusion The repaired tissue by tissue engineered cartilage with marrow mesenchymal stem cells as seed cells showed the best result in normal mechanical environment group, which indicates that it will be essential for the formation and maintenance of tissue engineered cartilage to keep the normal mechanical stress stimulus.
ObjectiveTo discuss the effectiveness of posterior cruciate ligament (PCL) reconstruction with autologous peroneus longus tendon under arthroscopy.MethodsBetween January 2016 and December 2018, 46 patients with PCL injuries were enrolled. There were 34 males and 12 females, with an average age of 40.7 years (range, 20-58 years). There were 43 cases of acute injury and 3 cases of old injury. The anterior drawer test and the posterior tibia sign were positive in 4 cases, the posterior drawer tests and the posterior tibia sign were positive in 46 cases, the varus stress tests were positive in 10 cases, and the valgus stress tests were positive in 6 cases. The difference of dial-test at 30° knee flexion between affected and healthy sides was (5.20±3.91)°. The tibia posterior displacement under posterior stress position was (12.03±2.38) mm. The Lysholm score of the knee joint was 36.68±7.89, the International Knee Documentation Committee (IKDC) score was 33.58±5.97, and the American Orthopaedic Foot and Ankle Association (AOFAS) score of the ankle joint was 97.60±1.85. PCL was reconstructed with autologous peroneus longus tendon under arthroscopy, and the combined meniscus injury, posterolateral complex injury, and anterior cruciate ligament injury were all treated according to the degree of injury.ResultsAll incisions healed by first intention. Forty patients were followed up 12-26 months, with an average of 16.0 months. At last follow-up, the Lysholm score of the knee joint was 84.85±7.03, and the IKDC score was 87.13±6.27, which were significant different from preoperative ones (t=?13.45, P=0.00; t= ?39.12, P=0.00); the AOFAS score of ankle joint was 93.98±2.14, which was not significant different from preoperative one (t=8.09, P=0.90). The tibia posterior displacement under posterior stress position was (2.75±1.76) mm and the difference of dial-test at 30° knee flexion between affected and healthy sides was (1.75±2.09)°, which were significant different from preoperative ones (t=29.00, P=0.00; t=4.96, P=0.00). The posterior drawer test and the posterior tibia sign were positive in 1 case and negative in 39 cases; the anterior drawer test and the varus and valgus stress tests were all negative.ConclusionReconstruction of PCL with autologous peroneus longus tendon under arthroscopy can significantly improve the stability and function of the knee joint, with satisfactory clinical results.
OBJECTIVE To study the feasibility of constructing tissue engineered cartilage by differentiated rabbit bone marrow mesenchymal stem cells(MSC) cultured in vitro and in vivo. METHODS The MSC were isolated from the nucleated cells fraction of autologous bone marrow by density gradient centrifuge, and then induced into chondrogenic differentiation by adding dexamethasone, transforming growth factor-beta 1 (TGF-beta 1) and ascorbic acid in vitro. After 3 weeks, some cells turned to round shape and secreted metachromatic matrix. The cartilaginoid grafts composed of chondrogenic MSC. Bovine type I collagen and human fibrin were cultured within the chondrogenic medium for 2 weeks in vitro or transplanted subcutaneously adjacent to the knee joint for 3 weeks in vivo. RESULTS The most cells in the grafts were degenerated and disappeared after cultured in vitro. But the residual cells were survival and secreted metachromatic staining proteoglycan with toluidine blue, which was characteristic cartilage matrix. The grafts developed into matured cartilage tissue assessed by histological examination after 3 weeks of transplantation in vivo. CONCLUSION MSC can be used as functional cells to constructing tissue engineered cartilage.
Acute kidney injury (AKI) is characterized by a sudden and rapid decline of renal function and associated with high morbidity and mortality. AKI can be caused by various factors, and ischemia-reperfusion injury (IRI) is one of the most common causes of AKI. An increasing number of studies found out that exosomes of mesenchymal stem cells (MSCs) could alleviate IRI-AKI by the adjustment of the immune response, the suppression of oxidative stress, the reduction of cell apoptosis, and the promotion of tissue regeneration. This article summarizes the effect and mechanism of MSC-derived exosomes in the treatment of renal ischemia-reperfusion injury, in order to provide useful information for the researches on this field.