Objective The key points in the epithelial-mesenchymal transition ( EMT) procedure include the downregula-tion of epithelial protein (E cadherin) and the upregulation of cell activity and cell matrix generation .The aim of this study was to es-tablish a method for primary culture and identification of mouse renal tubular epithelial cells and to explore whether the activation of C3aR can induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells . Methods Murine renal tubular seg-ments were used for primary cell culture .Immunocytochemistry and immunofluorescence staining were used to identify the renal tubular epithelial cells.The experiment groups included control group , five different concentrations of C3aR agonist groups (0.1, 1, 100, 500, and 2000 ng/mL), and three different time-point groups.The mRNA levels of E-cadherin,α-smooth muscle actin (SMA) and colla-gen I in renal tubular epithelial cells were detected by Real-time PCR; the protein of E-cadherin, α-SMA were detected by Western blot.The cytoskeleton of epithelial cells was observed by phalloidin staining . Results Compared with the control group , the protein expression of E-cadherin deceased (0.950±0.901 vs 0.650±0.221) and the expression of α-SMA (1.380±0.062 vs 1.600±0.103) and collagen I increased in C3aR agonist group (500 ng/mL, after 48 hours) (P<0.05).In addition, the association between these changes and C3aR agonists was presented in a dose-and time-dependent man-ner, respectively.The cytoskeleton staining showed that treatment of renal tubular epithelial cells with C 3aR agonists induced the formation of actin stress fibers in a time-dependent manner . Conclusion The method for primary culture and identification of mouse renal tubular epithelial cells were successfully established .The activation of C3aR could induce epithelial-to-mesenchymal transition in mouse primary renal epithelial cells , which plays an essential role in the de-velopment of renal fibrosis .Moreover , this study indicated that C 3aR may become a new therapeutic target in kidney diseases .

[Abstract ] Objective Evidence from previous studies indicated that over-activation of C3a/C3aR axis existed in the renal tubular epithelial cells of patients with renal diseases including diabetic nephropathy .However , the pathological significance of over-ac-tivating C3a/C3aR axis still remains to be elucidated .In this study, we constructed a renal tubular epithelial cell line over-expressing C3a in a secretory manner in order to provide a cell model to investigate the pathological significance of over -activating C3a/C3aR axis under various pathological scenes . Methods We designed a synthesized C3a secretory expression unit and cloned it into the multi-clonal site of lentivirus expression vector pLenti 6.3-MCS-IRES2-EGFP.After identification by sequencing , recombinant lentivirus was packaged by using pLenti 6.3-C3a-IRES2-EGFP and packaging plas-mid in 293T cells.Then, the recombinant lentivirus was used to in-fect HK2, a cell line of human renal tubular epithelial cells .After screening in medium with blasticidin , blasticidin resistant cell clones were obtained .Real-time PCR and ELISA method were applied to analyze the expression and secretion of stable transfected cells cloned C3a and identify renal tubular epithelial cell lines with stable over-activating C3a. Results ①C3a secretory expression unit was suc-cessfully synthesized and correctly cloned into the multi-clonal site of pLenti6.3-IRES2-EGFP; ②C3a secrectory expression recombi-nant lentivirus LV-C3a was successfully packaged with a high titer of 5 ×108/mL;③HK2 Cell clones resistant for blasticidin were ob-tained;according to the analysis of Real-time PCR and ELISA, the C3a mRNA level in HK2-C3a cell lines was significantly higher than that of HK2 cells(1.0 ±0.5 vs 1321.0 ±18.0, P<0.01) and the secreted C3a level increased significantly ([0.3 ±0.2]ng/mL vs [249.0 ±37.0] ng/mL, P<0.01). Conclusion The present study successfully constructed C 3a secretory expression vector pLenti6.3-C3a-IRES2-EGFP and C3a over-expression renal tubular epithelial cell line HK 2-C3a, which is very useful in further study of the function and significance of C 3a/C3aR axis not only in renal tubular epithelial cells but also in other cell types .

Objective Our previous study showed that the expression of C 3aR was increased in renal tubular epithelial cells (RTEC).However, the role of C3aR in diabetic nephropathy remains unknown, and so does the exact physiological and pathological signifi-cance of C3aR in relevant renal tissues.In the present study, we investigated the physiological and pathological significance of C3aR in RTECs using a lentivirus expression vector and anRTEC strain overexpressing C3aR. Methods Based on the sequence of C3aR mRNA, the human C3aR gene was synthesized and cloned into the multi-clonal site of the lentivirus expression vector pLenti6.3-MCS-IRES2-EGFP to construct a C3aR expression vector pLenti6.3-C3aR-IRES2-EGFP.After identification by sequencing, the recombinant lentivirus expressing C3aR was packaged by cotransfecting293T cellswith the recombinant lentivirusexpression vector and packaged plasmid .Then,the recombinant lentivirus was used to infect the human RTECline HK 2.After screening in the medium with blasticidin, blasticidinr-esistant cell clones were obtained, followed by identification ofthe human RTECline stably overexpressing C 3aR by real-time PCR and immunochemical staining. Results TheC3aR expression vector pLenti6.3-C3aR-IRES2-EGFP was successfully constructed and the sequence was proved to be correct. C3aR expression recombinant lentivirus was successfully packaged with a titer of 5×108/mL.Blasticidin-resistant HK2 cell clones were ob-tained and the expression of HK2-C3aR mRNA was significantly higherin the HK2-C3aR cells than in the non-transfected HK2 cells (2.33± 0.45 sv 1.00±0.09, P<0.05). Conclusion We successfully constructed a C3aR expression lentivirus vector pLenti6.3-C3aR-IRES 2-EGFP and a C3aR overexpression renal tubular epithelial cell strain HK2-C3aR, which is very contributive to further studies of the roles of C3aR in renal tubular epithelial cells and other types of cells.

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF ?1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF ?1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF ?1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF ?1 secretion in local site. [

0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group ( P0.05). There was not found no regular change of serum cytokine s (IL-6, TN F-?) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patien ts during post-operation from day 1 to day 30, indicating that was associated wi th the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involv ed in the repair of the injury induced by TNF-? in allo-transplanted livers.

Objective To explore the construction and application effects of extended care form for children with epilepsy based on Omaha System.Methods Based on Omaha System,the form was established by referring medical records,literature review and three rounds of focus group discussion.From February to June,2016,48 children with epilepsy were selected as the observation group and received routine care as well as management using the form based on Omaha System;from August to December,2015,48 children with epilepsy were selected as the control group and received routine care and follow-up.The effects of intervention and scores of Family Management Measure were compared between two groups.Results Nursing issues in all domains for the observation group 3 months after intervention were lower than those during hospitalization except financial situation (P＜0.05);there was statistically significant difference in scores of knowledge-behavior-status of main nursing issues before and after intervention except cognition and mental health items (P＜0.05).Duration of hospitalization,expenditure,readmission rate,EEG results and scores of FAMM in the observation group were better than those in the control group (P＜0.05).Conclusion The construction and application of the form based on Omaha System can provide references for longterm management for children.

Objective In this study ,we investigated the apoptosis of hair cell in the cochlea of age -related hearing loss(AHL) generated by ENU mutagenesis ,and to study a pan caspase inhibitor (z-VAD -FMK) which is to protect the cochlea hair cells from hearing loss induced by age-related hearing loss(AHL) .Methods Through z-VAD-FMK intraperitoneal injection and round window membrane (RWM) drug were delivered into the Cdh23 nmf308 nmf/nmf mice 5(postnatal days 2 -32) inner ear .ResuIts The results showed that the nmf308 mice with progressive hair cell loss along a base to apex gradient with age-related hearing loss .The cochlear OHCs reduced from 5% ~10% at 1 month to 100% at 3 month in the basal region .Substantial amounts of TUNEL -positive OHCs nuclei appeared at 1 month age ,and activated caspase-3 labeling demonstrated that most OHCs appeared at 2 months age .These suggested that DNA single strand break was attributed primarily to apoptosis of cochlear le_sions ,whereas in the later stage of lesion ,the expansion led to activation of caspase-3 activity reduced with further progression of nuclear condensation in age-related hearing loss .ConcIusion The addition of a pan caspase inhibitor (z -VAD -FMK ) significantly protected the cochlea against the hair cell loss induced by apoptosis .Our study showed that aspase inhibitor ,Z-VAD-FMK appeared to play a prominent role in age-related hearing loss media_ted hair cell death loss induced by apoptosis .Our study showed that aspase inhibitor ,Z-VAD -FMK appeared to play a prominent role in age-related hearing loss mediated hair cell death .

Objective To explore the medial open wedge high tibial osteotomy (OWHTO) combined with arthroscopic condyle plasty with clinical flexion deformity in patients with osteoarthritis of knee varus.Methods From January 2014 to July 2015,11 cases (11 knees) of varus knee joint flexion deformity were applied the procedure of OWHTO combined with arthroscopic condyle plasty,including 4 male and 7 female;the average age was 52.1 years old (ranged from 48 to 58 years).OWHTO could adjust the line of force of the lower limbs in coronal plane (the connection line between femoral head center point and the center point of ankle joint) through lateral tibial plateau 62.5％ position,and implant allogeneic bone to support posterior inclination angle,underwent arthroscopic condyle plasty to improve flexion deformity at the same time.Results 11 patients in this group were all followed up,and the follow-up time was 1-2.5 years,average 1.5 years.No case of fracture nonunion or delayed healing was found.Imaging data was measured to evaluate the mechanical axis of lower extremity by the relative position of tibial plateau,femoral tibial angle,femoral notch width index,tibial plateau posterior angle before operation and one year after the operation.The relative position of the mechanical axis of the lower limb through the tibial plateau was 17.4％ ± 4.9％ preoperatively,and 58.9％ ± 3.1％ after operation,femoral tibial angle changed from 181.6°± 1.2° to 170.3°± 1.3°,tibial plateau posterior inclination angle:preoperative 7.7°±2.2°,postoperative 7.9°±1.9°,femoral notch width index was increased from 0.221±0.007 to 0.272±0.009 after operation,flexion deformity angle of preoperative was 11.1 °± 3.1 °,and 1.4°± 1.5° one years after operation,VAS score was (6.5 ± 1.1)points before surgery,and (2.5±0.8) points of postoperative,Lysh(o)lm score was (50.72±6.57) points before operation,and (75.72±7.41) points one year after operation,and the differences were all statistically significant.Conclusion 0WHT0 combined with arthroscopic condyle plasty can significantly improve the lower limb line and flexion deformity,and also maintain the posterior inclination angle of tibial plateau,and can get a good short-term efficacy.

Aim To investigate the protective effects of dihydroquercetin(DDQ) against myocardial ischemis reperfusion injury(MIRI) in rats.Methods Male Sprague-Dawley rats were randomly divided into 4 groups(n=10):normal,control,I/R model, and I/R model+DDQ(5,10 mg·L-1).This study used an isolated Langendorff rat heart model.The left ventricu-lar developed pressure(LVDP),heart rate(HR) and the maximum rise and fall rate of the left ventricular pressure(±dp/dtmax) were monitored and documented using a physiological recorder.The levels of lactate dehydrogenase(LDH) and creatine kinase(CK) were analyzed using enzyme-linked immunosorbent assay(ELISA).Infarct size was measured using 2,3,5-triphenyltetrazolium chloride staining.The levels of superoxide dismutase(SOD) and malondialdehyde(MDA), as well as the ratio of glutathione/glutathione disulfide(GSH/GSSG) were measured via ELISA.HE staining was used to observe the pathological changes of myocardial tissue.Results Compared with the I/R model group, the I/R model+DDQ groups raised hemodynamic parameters, SOD level, and GSH/GSSG ratio;and reduced the amount of CK, LDH, MDA levels.Moreover, the I/R model+DDQ groups had lower infarct size and pathological changes in myocardial tissue than I/R model group.Conclusion DDQ exertes cardioprotective effects against I/R via improving the oxygen free radical scavenging ability, the inhibition of oxygen free radical and reducing lipid peroxidation.

Objective To investigate the correlation of medial meniscus extrusion with meniscus injury location, type and genu varum. Methods From March 2014 to March 2015, a total of 97 patients with injury of medial meniscus undergoing ar?throscopy and surgery were selected as subjects, including 40 males and 57 females, aged 32-66 years, with a mean age of 51.2± 10.71 years. Based on the MRI of knee, there were 38 cases with medial meniscus extrusion≥3 mm (extrusion group) and 59 cases without medial meniscus extrusion (non?extrusion group). Genu varus was measured on X?ray (Femur?Tibia?Angle<182°). The me?dial meniscus tear type was observed under arthroscopy (longitudinal tear, horizontal tear, oblique tear, radial tear, complex tear), as well as the meniscus tear location (anterior tear, body tear, posterior tear, root tear). The age, gender, BMI and involved side were compared between two groups. Multifactor unconditioned Logistic regression analysis was employed to analyze risk factors of medial meniscus extrusion. Results Two groups of patient showed no statistically significant difference in age (t=-1.511, P=0.135), gender (χ2=0.208, P=0.648), BMI (t=0.249, P=0.650) and side (χ2=0.052, P=0.820). The incidence of meniscus extru?sion in patients with genu varum was 89% (25/28), and the incidence of meniscus extrusion in patients with meniscus root tear was 78% (18/23). Significant difference could be spotted in the analysis of meniscal root tear (χ2=19.329, P=0.000), complex tear (χ2=5.111, P=0.024), genu varus (χ2=41.481, P=0.000) between patients with medial meniscus extrusion or without medial meniscus extrusion. Meanwhile, meniscus anterior tear (χ2=0.044, P=1.000), body tear (χ2=0.261, P=0.661), posterior tear (χ2=3.722, P=0.086), longitudinal tear (χ2=0.054, P=0.816), horizontal tear (χ2=0.317, P=0.790), oblique tear (χ2=0.198, P=0.819), radial tear (χ2=1.188, P=0.385) no statistical significance. By multifactor analysis, OR values of genu varus and root tear were 101.976 (95%CI:15.973, 651.041, P=0.000) and 35.517 (95%CI:6.804, 185.399, P=0.000), respectively. Conclusion Menis?cal root tear and genu varum were risk factors of medial meniscus extrusion.