Objective To analyze the effect of GC-rich DNA fragments on the level of transgenic expression in Chinese hamster ovary (CHO) celts and its position effect.Methods The synthetic DNA fragment with GC-rich was cloned into the 5'or 3'or both 5'and 3'ends of expression cassette of expression vector.Three new expression vectors (pIRES-G1,pIRES-G2 and pIRES-G3) which was inserted with the GC-rich DNA fragments in different position were transfected CHO ceils,respectively,and then was observed under fluorescence microscope;the control vector was pIRES-EGFP.Stable transfected cell lines were screened under G418,and enhanced green fluorescent protein(EGFP) expression was analyzed by flow cytometry and the transgenic copy number was detected by quantitative real-time quantitative polymerase chain reaction (qRT-PCR).Results Three expression vectors with a GC-rich DNA fragments in different position were constructed successfully.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector could obviously improve the expression level of vector in CHO cells;and the expression level of the stably transfected CHO cells increased 1.39 fold and 1.32 fold compared to the control vector,respectively;the transgene copy number increased 1.32 fold and 1.24 fold compared with the control vector.While the insertion of GC-rich DNA fragments at 5'end of expression cassette had no obvious effect on the level of gene expression.Conclusion The role of DNA fragment with GC-rich in improving the transgenic expression of CHO cells is related to its position in the vector.The insertion of GC-rich DNA fragments at 3'end and both 5',3'ends of the box of expression vector can improve transgenic expression.

0.05).The ADCs of low-grade(gradeⅠ～Ⅱ) astrocytomas were significantly higher(P

Background Retinal pigment epithelial (RPE) cell transplantation is a novel approach to the treatment of hereditary retinal diseases, however, human-derived RPE cell line occurs de-differentiation during in vitro cell culture.Studies showed that early abnormal activation of Akt/mammalian target of rapamycin (mTOR) signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of mTOR pathway will be helpful for the retard of de-differentiation of RPE cells.Objective This study aimed to investigate whether rapamycin can suppress the activation of mTOR pathway and promote differentiation of ARPE19 cells.Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group.DMSO or rapamycin with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamycin-treated group, respectively.The cells of each group were collected 24 hours and 48 hours after cultured.The expression of zonula occludens-1 (ZO-1) in the cells was examined by immunofluorescence.The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot, respectively.The detected results were compared between the two groups.Results ZO-1 was expressed in both group,but the fluorescence intensity was evidently enhanced in the rapamycin-treated group.The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97％ , 29.71％ and 13.00％ in the rapamycin treated group compared with the control group 24 hours after cultured (P=0.04,0.04,0.04) , and the expression levels of RPE65, LRAT, rLBP1, BEST1 , keratin18 and MERKT mRNA elevated by 174.00％ , 88.00％ , 56.18％ ,193.81％ ,10.83％ and 35.02％ in the rapamycin-treated group in comparison with the control group 48 hours after cultured (P =0.00,0.04,0.01,0.04,0.04,0.03).In addition, the expressions of p-mTOR, p-P70S6 and p-S6 protein were weaker in the rapamyein-treated group than those in the control group both 24 hours and 48 hours after cultured.Twenty four hours after cultured,the expression level of ZO-1 protein raised by 40％ in the rapamycin-treated group compared with the control group (P =0.01);while 48 hours after cultured,the expression levels of ZO-1 ,MERKT, catenin and LRAT proteins elevated by 36.00％ ,57.37％, 13.68％ and 41.07％ in the rapamycintreated group in comparison with the control group (P=0.01,0.00,0.04,0.04).Conclusions Rapamycin can suppress the activation of mTOR signaling pathway and up-regulate the expressions of RPE specific genes in ARPE19 cells.Inhibition of mTOR pathway might be an effective way for culturing RPE cells in vitro.

Objective: To understand the attitudes and perceptions of traditional Chinese medicine (TCM) practitioners in Beijing TCM hospitals regarding the use of Ephedra sinica Stapf (E. sinica, Ma Huang). Methods: A two-stage cross-sectional study was conducted using a questionnaire survey of TCM practitioners in Beijing TCM hospitals between April 2023 and March 2024. The questionnaire included demographic information, the clinical background of TCM practitioners, and the clinical application of E. sinica. Logistic regression analysis was used to analyze the relevant influencing factors when using E. sinica. Results: Of the 465 questionnaires collected, 441 were valid. Among these, 84.81% (374/441) reported having used E. sinica in clinical practice at least once. The commonly used doses of E. sinica—excluding the pediatric department—were 10 g for high doses, 6 g for medium, and 3 g for low. The three most frequently used formulas for E. sinica included Maxing Shigan decoction, Mahuang decoction, and Xiao Qing Long decoction. The most common TCM patterns treated with a high dose of E. sinica were wind-cold exterior pattern, wind-cold invading the lung, and wind and water combat with meridians congealed by cold. The top three Western medical diagnoses when using E. sinica for treatment were common cold, pneumonia, and upper respiratory tract infections. Nearly half of the respondents reported experiencing adverse reactions from the oral administration of E. sinica, with the most common being palpitations, insomnia, and restlessness. Starting with a low dose and gradually increasing it as appropriate was identified as an effective approach. Conclusion This study investigated the attitudes and perceptions of TCM practitioners in Beijing TCM hospitals regarding the dose–efficacy–adverse reaction relationship of E. sinica, providing a reference for the safe and effective clinical use of E. sinica.

Objective To analyze the effect of using standardized patient (SP) tutorial in the spe-cialized training of pancreatic surgery with WeChat platform. Methods 48 surgeons participating in resi-dent standardized training in Changhai Hospital (all for postgraduate education) were enrolled as teaching object. 48 surgeons were divided into two groups: SP group (n=24) receiving WeChat combined with SP tutorial which updates learning plan, learning contents, and clinical discussion by Wechat platform and performs practical teaching by SP method, control group (n=24) receiving traditional tutorial by using tradi-tional clinical teaching methods and video teaching followed by practical teaching. The theoretical exami-nation, questionnaires and expert assessment were used to evaluate the effect of the two teaching methods. Statistical analysis was performed using the SPSS 19.0. Continuous data were expressed as median±stan-dard deviation and compared using the Student's t-test. Categorical data were compared using the Pearson's chi-square test. Results The score of theoretical examination of the two groups showed no significant difference [(85.5±7.6) vs. (81.4±14.9), P=0.238]. The results of questionnaires and expert assessment in WeChat&SP group were significantly better than those in the control group (P<0.001) other than theoretical and analytical ability (P>0.05). Conclusion WeChat platform combined with standardized patient tutorial in the specialized training of pancreatic surgery is feasible and more effective than traditional tutorial to improve teaching effectiveness.

Objective:To investigate the effect of BMSC transplantation on the recovery of neurological function in rats with cerebral infarction,and to explore the related mechanism.Methods:90 rats were randomly divided into 3 groups:sham operation group,control group,BMSC transplantation group,30 rats in each group.The control group and BMSC transplantation group established middle cerebral artery occlusion (MCAO) model,the sham operation group only need to separate the cervical tissue of rats,and MCAO model in the MCAO model operation.After 1 days of BMSC transplantation group by intravenous injection of 1 mL 3× 106 BMSC,the control group was injected with the same dose of NS in MCAO after 1 D,3 D,7 d,14 d,21 d,28 d,35 d,42 d,49 D respectively,the neurological function score of rats (mNSS),after 2 months of transplantation BMSC group and control group of brain tissue for immunohistochemical staining,detection of MAP2,TUJ1,Ⅷ factor,the expression of GFAP.Results:In seventh to thirty-fifth days after treatment,BMSC mNSS transplantation group were significantly lower than the control group (P ＜ 0.05).2 months after BMSC transplantation group MAP2,TUJ1,Ⅷ expression level was significantly higher than the control group,while the control group,the expression of GFAP was significantly higher than that of BMSC group (P ＜ 0.01).Conclusion:BMSC transplantation in order to promote the recovery of neurological function in cerebral infarction.

Objective:To explore the influence of rosuvastatin in the lipid levels, atherosclerosis plaque and apoptosis in the plaque of atherosclerotic apolipoprotein E (ApoE)-knockout mouse models, and to clarify the mechanism of rosuvastatin in inhibiting atheromatous plaque and apoptosis in the plaque.Methods:Thirty healthy six-week old male mice were randomly divided into high fat diet group (n=10),rosuvastatin group (n=10)and general diet control group (n=10).The mice in first two groups were fed with high fat diet,and the mice in general diet control group were fed with general diet;4 weeks later the mice in three groups were respectively given 0.9% NaCl solution,rosuvastatin (10 mg·kg·d-1 )and 0.9% NaCl solution by gavage for 8 weeks.And then ELISA was used to detect the serum lipid levels,the area of atherosclerotic plaque was measured by HE staining, and the apoptosis in plaques was detected by TUNEL method;the expression of apoptosis-assoicated gene Bax protein was measured by immunohistochemical staining.Results:Compared with high fat diet group,the levels of total cholesterol (TC)and low density lipoprotein cholesterol (LDL-C)of the mice in rosuvastatin group were significantly decreased (P<0.05 or P<0.01),and the 1evel of high density lipoprotein cholesterol (HDL-C)was significantly increased (P<0.01).The aortic atherosclerotic plaque of the mice in high fat diet group was massive with a great quantity of foam cells, cholesterol crystal, necrotic cells and inflammatory cells in the plaque;the aortic atherosclerotic plaque of the mice in rosuvastatin group was smaller with less foam cells,necrotic cells and inflammatory cells;the aortic atherosclerotic plaque area of the mice in rosuvastatin group was significantly smaller than that in high fat diet group (P<0.01);the apoptotic index in the aortic atherosclerotic plaque of the mice in rosuvastatin group was significantly lower than that in high fat diet group (P<0.01).Compared with high fat diet group,the expression of Bax protein in rosuvastatin group was significantly decreased.Conclusion:Rosuvastatin may inhibit the progression of atherosclerotic plaque and the apoptosis in plaques through mitochondrial pathway.

Objective To analyze the clinicopathologic data and operative parameters of 209 elderly co-lon cancer patients treated by radical resection between January 2002 and December 2011 ,and to investigate the factors related to recurrence and metastasis after colon cancer radical resection in elderly patients .Methods We used univariate and multivariate analysis of Cox regression ,including 14 variables:age,gender,disease duration, hospitalization duration,surgeon experience,operation duration,laparoscopicsurgery,tumor location,tumor size, gross morphology ,differentiate degree ,depth of bowel wall invasion ,lymph node involvement and obstruction .The survival curve was obtained by Kaplan -Meier method.Results Univariate analysis showed that tumor size (RR=2.658,P<0.0001),gross morphology(Infiltrating type,RR=3.407,P=0.0054),degree of differentiation (RR=0.32,P<0.0001) were associated with tumor relapse and metastasis .Multivariate analysis showed that gender(RR=0.585,P=0.0359),tumor size(RR=2.364,P<0.0001),degree of differentiation (Infiltrating type,RR=0.246,P=0.0437),gross morphology(RR=0.31,P<0.0001)were the significant factors.Conclu-sion Gender,tumor size,degree of differentiation,gross morphology were the independent factors of recurrence and metastasis of colon cancer after radical resection in elderly patients .Targeted follow -up for high -risk groups will improve patients′life quality and prolong their survival time .

Objective To investigate the effect of high mobility group box chromosomal protein 1 (HMGB-1) on the proliferation and inflammatory phenotype of human fibroblast like synoviocytes (FLS).Methods FLSs were isolated from the synovial tissues of rheumatoid arthritis (RA) patients undergoing joint replacement surgery.All the experiments described here utilize the FLSs between the third and sixth passages.FLS were incubated with HMGB1 at (100,500,2 000 ng/ml) or interleukin (IL)-1β (0.5 ng/ml) or lipopolysaccharide (LPS) (100 ng/ml) alone or HMGB1/IL-1β complexes or HMGB1/LPS complexes.Cell proliferation assay were used by CCK-8,IL-6,IL-8 and matrix metalloproteinase-3 (MMP-3) in culture supernatants were measured using enzyme-linked immunosorbent assays.The measurement data were compared with single factor analysis of variance.Results Stimulation with all concentrations of HMGB1,IL-1β and LPS alone did not affect the cell proliferation of FLS.HMGB1/IL-1β complexes and HMGB1/LPS complexes did not affect the cell proliferation of FLS,neither (F=0.415.P=0.915).Except high concentration of HMGB1 (2 000 ng/ml) could significantly stimulate the secretion of IL-6 from FLS [(23.0±1.1) ng/ml],HMGB1,IL-1β and LPS alone did not affect the production of IL-6,IL-8 and MMP-3.However,HMGB1/IL-1β complexes and HMGB1/LPS complexes increased IL-6,IL-8 and MMP-3 production from FLS (F=97.804,117.383,70.179,P=0.000).Conclusion Neither HMGB1,IL-1β,LPS alone nor HMGB1/IL-1β complexes or HMGB1/LPS complexes affect the cell proliferation of FLS.HMGB1 in complex with LPS or IL-1β boost IL-6,IL-8 and MMP-3 production in synovial fibroblasts from RA patients.

Background Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition,iPS-RPE cells may provide a personalized treatment platform for the patient's own cells treatment.Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes.Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p.S1087L and individual without SNRNP200 p.S1087L mutation,respectively,with the size 2 c m×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cellconditioned medium to generate and induce iPSCs,and then the iPSCs were identified by morphology,alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65,zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement,and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation,and the positive expressions of hESC-specific surface antigens including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation,and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65,ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.Conclusions Mutation SNRNP200 p.S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.