BACKGROUND: Both density gradient centrifugation and adherence method arc frequently used to isolate bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the approaches to isolate, culture and identify the rabbit BMSCs in vitro by the combination of den,ity gradient centrifugation and adherence method. DESIGN, TIME AND SETTING: Contrast cytological study, which was performed in Central Laboratory of Shanghai 6th People's Hospital between October 2007 and March 2008.MATERIALS: Six 2-week-old rabbits were selected for BMSCs preparation and primary culture; Percoll separating medium (1.073 kg/L) was also used for this study.METHODS: BMSCs were separated and purified with Percoll separating medium by density gradient centrifugation and adherence method. The three-, five-, seven-, and nine-passage BMSCs were counted for growth curve. MAIN OUTCOME MEASURES: Morphological features and growth states of primary and passage cells were observed under inverted microscope. Indirect immunofluorescence of CD44 and CD34 antibodies were used to examine the stem cells. CD44 staining was positive, and CD34 staining was negative, suggesting the extracting and purifying cells were BMSCs. RESULTS: The passage BMSCs were uniformly distributed like fusiform shape, which were more uniform than primary cultured cells. The BMSCs grew productively and proliferated rapidly; meanwhile, the nucleolus was clear, caryopla.sm was in a large proportion, morphological features were uniform, ceils like bostrychoid or whirlpool were arranged parallelly, and the five-pa.ssage cells were not changed remarkably. Proliferation was decreased gradually with the passage increasing; especially, the proliferation of three-five-passage cells was the strongest. The separated cells expressed CD44 but not CD34. CONCLUSION: High-purified rabbit BMSCs are obtained by both density gradient centrifugation and adherence method.

Objective:To investigate the effect of repetitive transcranial magnetic stimulation (rTMS) on cognitive function in patients with chronic schizophrenia.Methods:100 cases patients with chronic schizophrenia weree selected.They were divided into rTMS stimulation group and pseudo stimulation group according to the random number table method,and each group include 50 cases.The positive and Negative Syndrome Scale (PANSS) and the Treatment Emergent Symptom scale (TESS) were used to assess the clinical symptoms and side effects before and after the treatment;The Wisconsin Card Sorting Test (WCST) and Repeatable Neuropsychological State Measurement (RBANS) were used to assess the patients' cognitive function before and after treatment.Results:After treatment,the PANSS total score,the positive subscale score,the negative subscale score,the general psychopathological subscale score in the rTMS stimulation group were significantly reducing (P＜0.05),and significantly lower than those in the pseudo stimulation group (P＜0.05),but the TESS scores between the two groups did not differ significantly before and after treatment.The percentage of WCST in rTMS stimulation group was significantly higher than that in the pseudo stimulation group (P＜0.05),and the total time and wrong thinking time was shorter than that in the pseudo stimulation group (P＜0.05);The visual span and delayed memory performance of RBANS was significantly increased in the rTMS stimulation group (P＜0.05),and the visual span was significantly higher than that in the pseudo stimulus group (P＜0.05);There were 2 cases of patients with mild symptoms after the first treatment in RTMS stimulation group,all patients were without complaint followed up for 3 months.Conclusion:rTMS treatment on the part of the cognitive function of patients with chronic schizophrenia has a certain improvement effect,and higher security,it is worth further study.

Objective This study investigates feasibility of replacing urinary epidermal cells with foreskin epidermal cells to reconstruct engineered anterior urethra with acellular collagen matrix. Methods A cellular collagen matrices were generated from allogeneic rabbit bladder submucosa.In 6 rabbits,autologous foreskin epidermal cells isolated,in vitro expanded and labeled with Brdu before seeding onto a tubular collagen matrix of 2 cm long.In 12 male rabbits, urethral mucous defect was created and urethroplasty was performed with tubular collagen matrix seeded with epidermal cells (n=6) in experimental group or matrix without cells seeding (n=6) in control group.Urethrography was performed at 1,2,and 6 months postoperatively.Urethras grafts were harvested and analyzed grossly and histologically at the same time points. Results In control group, gross and urethrography demonstrated urethral stricture of repaired defects at all time points.Histology showed a single layer of epidermal cell with unorganized and crassitude muscle fiber bundles in submucosa layer.In contrast, urethrography showed a patency of repaired urethral defect with the maintenance of a wide urethral caliber in experimental group without sign of strictures at any time points. Histologically,the graft seeded with epidermal cell had a multi-layers epidermal cells by 1 month,which demonstrated the presence of a epidermal cells layer with a normal submucosa structure.The immunoflorescent staining of Brdu confirmed the presence of implanted epidermal cells.In addition, the implanted cells also express keratin when stained with anti-pancytokeratins AE1/AE3 antibody.At 2 month,histology showed relatively normal urethral architecture with vessel abundance in submucosa structure.The staining of Brdu showed decreased at implanted epidermal cells and epithelial phenotypes were confirmed immunocytochemically using pancytokeratins AE1/AE3.At 6 month,the graft structure show multi-layers epidermal cells without staining of Brdu. Conclusions Urethra reconstruction was better achieved with cell seeded acellular collagen matrix when compare with acellular matrices alone.Foreskin epidermal cells seems succeeded in replacing urethral epidermal cells for urethra reconstruction.

Objective:To effectively express the receptor binding domain (RBD) of SARS-CoV-2 spike protein in Pichia pastoris and to evaluate its immunogenicity. Methods:The gene encoding the RBD protein was synthesized and cloned into the pPICZαA plasmid. After linearization, the plasmid was transferred and integrated into the genome of Pichia pastoris. The expressed RBD protein in culture supernatant was analyzed by Western blot and Biolayer interferometry. After screening, a single clone expressing the RBD protein was selected. The high-level expression of RBD protein was achieved by optimizing the fermentation process, including the salt concentration adjusting of the medium and induction condition optimization (pH, temperature and duration). The immunogenicity of the expressed RBD protein was evaluated in a mouse model. Results:A single clone with a high expression level of RBD protein was obtained and named RBD-X33. The expression level of RBD protein in the fermentation supernatant reached up to 240 mg/L after optimization of the induction condition (HBSM medium, pH=6.5±0.3, 22℃ and 120 h). In the mouse experiment, the recombinant RBD protein was formulated with Alum+ CpG dual adjuvant and injected into mice. The binding IgG antibody levels were up to 2.7×10 6 tested by ELISA and the neutralizing antibody levels were up to 726.8 tested by live virus neutralizing antibody assay (prototype). Conclusions:The RBD protein could be efficiently expressed in Pichia pastoris and induce stronger immune response in animals. This study suggested that the recombinant SARS-CoV-2 RBD protein expressed in Pichia pastoris could serve as a candidate antigen in the development of SARS-CoV-2 vaccine.

Microbial Engineering is a practical and applied course, it is therefore an indispensable part of teaching to let the students practice in the production line of an enterprise. However, lack of internship resources has always been a bottleneck hampering the high-level internship training during teaching the Microbial Engineering course. A virtual production line developed based on information technology enables students to learn the theoretical knowledge of the complex production system. Moreover, it enables students to perform complex virtual operations, thereby improving students' practicing and innovation abilities. In order to let the virtual simulation experiments project play a great role in undergraduate teaching, this article summarizes the progress, characteristics, and value of the virtual simulation experiments for Microbial Engineering. In addition, the main challenges that limit its shared application are also discussed, with potential solutions prospected.
Computer Simulation ; Curriculum ; Humans ; Learning ; Students

Osteoporosis （OP） is a systemic metabolic bone disease. Amid population aging， OP has become a major health problem for the middle-aged and the elderly in China. Aging， iron load， and estrogen deficiency break the balance between oxidation and antioxidant systems， and the increase of reactive oxygen species mediates oxidative stress to damage DNA， lipids， proteins and other macromolecules， thus accelerating cell apoptosis and inducing OP， obesity， and neurodegenerative disorders. It has been found that oxidative stress is of great significance in the pathogenesis of OP. Oxidative stress regulates the signaling pathways， cytokines， and proteins related to the mesenchymal stem cells， osteoblasts， and osteoclasts， thereby weakening the osteogenic differentiation of mesenchymal stem cells， inhibiting osteoblast mineralization， and promoting the activation， proliferation， and maturation of osteoclasts. As a result， the dynamic imbalance between bone resorption and bone formation occurs， influencing bone remodeling and promoting the progression of OP. At the moment， anti-bone resorption drugs， bone formation-promoting drugs， and hormones are mainly used in clinical settings in western medicine. However， due to the long treatment cycle and the occurrence of serious gastrointestinal reactions， hypocalcemia， osteonecrosis， and others， patients show poor compliance and thus the effect is not as expected. Traditional Chinese medicine （TCM） demonstrates remarkable effect on OP attributing to the multi-pathway and multi-target characteristics. With low price and few adverse reactions， TCM is widely applied in clinical practice in comparison with western medicine. TCM has unique advantages in the treatment of OP by regulating oxidative stress. It exerts the therapeutic effect on OP by modulating different signaling pathways， providing new mindset for the treatment of this disease. Therefore， through literature research， this study summarized the research on mechanism of oxidative stress in OP and the treatment by TCM， which is expected to lay a foundation for further research.

Idiopathic pulmonary fibrosis （IPF）， as a progressive lung disease， has a poor prognosis and no reliable and effective therapies. IPF is mainly treated by organ transplantation and administration of chemical drugs， which are ineffective and induce side effects， failing to meet the clinical needs. Therefore， developing safer and more effective drugs has become an urgent task， which necessitates clear understanding of the pathogenesis of IPF. The available studies about the pathogenesis of IPF mainly focus on macrophage polarization， epithelial-mesenchymal transition （EMT）， oxidative stress， and autophagy， while few studies systematically explain the principles and links of the pathogeneses. According to the traditional Chinese medicine theory， Qi deficiency and blood stasis and Qi-Yang deficiency are the key pathogeneses of IPF. Therefore， the Chinese medicines or compound prescriptions with the effects of replenishing Qi and activating blood， warming Yang and tonifying Qi， and eliminating stasis and resolving phlegm are often used to treat IPF. Modern pharmacological studies have shown that such medicines play a positive role in inhibiting macrophage polarization， restoring redox balance， inhibiting EMT， and regulating cell autophagy. However， few studies report how Chinese medicines regulate the pathways in the treatment of IPF. By reviewing the latest articles in this field， we elaborate on the pathogenesis of IPF and provide a comprehensive overview of the mechanism of the active ingredients or compound prescriptions of Chinese medicines in regulating IPF. Combining the pathogenesis of IPF with the modulating effects of Chinese medicines， we focus on exploring systemic treatment options for IPF， with a view to providing new ideas for the in-depth study of IPF and the research and development of related drugs.