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.
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.
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.
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.
Superficial temporal artery (STA) - middle cerebral artery (MCA) bypass surgery has been widely used to treat patients with moyamoya disease, and its application value in symptomatic internal carotid artery (ICA)/MCA stenosis/occlusion remains controversial. With the development of imaging, micro-devices and surgical techniques, and the deepen understanding of diseases, the effectiveness of STA-MCA bypass surgery in the treatment of symptomatic ICA/MCA stenosis/occlusion is further required. This article reviews the process of development and evolution of this surgical technique, as well as the significance and deficiencies of several randomized controlled trials of ICA/MCA treatment in the past, and looks forward to possible improvements in future research, so as to clarify the way for further randomized controlled study.
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.
Objective To review research advances of revision surgery after primary total hip arthroplasty (THA) for patients with Crowe type Ⅳ developmental dysplasia of the hip (DDH). Methods The recent literature on revision surgery after primary THA in patients with Crowe type Ⅳ DDH was reviewed. The reasons for revision surgery were analyzed and the difficulties of revision surgery, the management methods, and the related prosthesis choices were summarized. Results Patients with Crowe type Ⅳ DDH have small anteroposterior diameter of the acetabulum, large variation in acetabular and femoral anteversion angles, severe soft tissue contractures, which make both THA and revision surgery more difficult. There are many reasons for patients undergoing revision surgery after primary THA, mainly due to aseptic loosening of the prosthesis. Therefore, it is necessary to restore anatomical structures in primary THA, as much as possible and reduce the generation of wear particles to avoid postoperative loosening of the prosthesis. Due to the anatomical characteristics of Crowe type Ⅳ DDH, the patients have acetabular and femoral bone defects, and the repair and reconstruction of bone defects become the key to revision surgery. The acetabular side is usually reconstructed with the appropriate acetabular cup or combined metal block, Cage, or custom component depending on the extent of the bone defect, while the femoral side is preferred to the S-ROM prosthesis. In addition, the prosthetic interface should be ceramic-ceramic or ceramic-highly cross-linked polyethylene wherever possible. Conclusion The reasons leading to revision surgery after primary THA in patients with Crowe type Ⅳ DDH and the surgical difficulties have been clarified, and a large number of clinical studies have proposed corresponding revision modalities based on which good early- and mid-term outcomes have been obtained, but further follow-up is needed to clarify the long-term outcomes. With technological advances and the development of new materials, personalized prostheses for these patients are expected to become a reality.
Objective To improve the knowledge of lung injury induced by rituximab. Methods Clinical data of 5 lymphoma patients with lung injury caused by rituximab chemotherapy were analyzed. Results Five patients received chemotherapy including rituximab, and had fever, cough and dyspnea after 3 to 5 chemotherapy cycles. Chest CT showed bilateral diffuse interstitial infiltrates. All 5 cases experienced hypoxemia or respiratory failure. Clinical symptoms were improved 3 to 5 days after the treatment of glucocorticoids, and pulmonary lesions were significantly alleviated 1 to 2 weeks after the treatment. According to the literature, the incidence rate of lung injury caused by rituximab was 0. 03% to 4. 9%, which has increased recently. Conclusions With the comprehensive application of rituximab, lung injury caused by this drug is not rare. The good prognosis depends on early diagnosis and treatment by further recognition of the side effect of rituximab.
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。
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.