Objective To establish a co-culture system between mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs) in vitro, and study the influence of SMCs on the differentiation of bone marrow MSCs (BMSCs) into SMCs in the co-culture system. Methods Density gradient centrifugation was used to separate and culture BMSCs, and collagenase digestion was employed to separate and culture bladder smooth muscle cells(BSMCs). Then the 2 types of cells were identified respectively. Then the cells were divided into 4 groups, BMSCs culture group (positive control), BSMCs cells culture group (negative control), BMSCs and BSMCs co-culture groups with contact or non-contact. The expression of ?-smooth muscle actin (?-SMA) and calponin were detected by immunofluorescence staining. Western blot analysis was employed to determine protein expression of calponin. Results In contact co-culture group, the positive rate of CD90 abd CD44 were 99.78% and 60.27% respectively in BMSCs, while only 0.21% for CD45. Western blotting showed that BMSCs expressed little calponin, but those contacting with BSMCs had an increasingly stronger expression of calponin with the elapse of contact culture time, while there was no significant change in its expression in BMSCs with contact. Immunofluorescence staining indicated that part of BMSCs were calponin positive at the 8th day after culture, but no positive cell was observed in non-contact culture group and BMSCs culture group. Conclusion The contact between BSMCs and BMSCs can promote the differentiation of BMSCs into SMCs.

Objective To study the effects of muscular trumatic fluid on the biological properties of muscle-derived stem cells (MDSCs) in rats.Methods MDSCs were isolated and purified by the preplate technique,muscle injury was made for the extraction of muscular traumatic fluid.Bradford colorimetric was used to detect the protein content in the muscular traumatic fluid,and the fluid with the highest protein was used to co-culture with MDSCs.Methyl thiazolyl tetrazolium (MTT) assay and wound model of monolayer of cells were used to observe the effects of wound fluids on the proliferation and mobility of MDSCs.The expressions of α-SMA and Vimentin were tested by immunohistochemistry and Western blot technique.Results MTT assay showed that wound fluid with concentration of 10% could most significantly increase the proliferation of MDSCs.The increased expressions of α-SMA and Vimentin were found in MDSCs after cultrue with muscular traumatic fluid in vitro,and the time-dependent relation exists.Conclusions Wound environment can directly participate in the muscle regeneration by inducing the proliferation,mobility of MDSCs;on the other hand,MDSCs can differentiate into fibrotic cells under stimulation of muscular traumatic fluid.

Objective To construct a lentiviral vector expressing small-hairpin RNA(shRNA) targeting Smad3 gene.Methods The targeting sequence of Smad3 gene which can be effectively silenced in RNA inference was confirmed in our previous study.The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed,synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing Smad3 shRNA was confirmed by PCR and sequencing.293T cells were cotransfected with lentiviral vector GC-shSmad3,pHelper 1.0 and pHelper 2.0.The titer of virus was tested according to the expression level of GFP.Results PCR and DNA sequencing demonstrated that the constructed lentivirus vector GC-shSmad3 produced Smad3 shRNA.The titer of concentrated virus was 3?108 TU/ml.Conclusion The lentivirus RNAi vector targeting Smad3 is constructed successfully.

Previous studies have suggested that various kinds of inflammatory factors can influence the formation and development of tumor cells.Researche has shown that gallbladder cancer is closely linked with local inflammation,which is a risk factor for the development of gallbladder cancer.It is widely known that cholecystitis is closely correlated with gallstones,and that bile obtained from patients with gallbladder cancer contains a large variety of bacteria,such as Salmonella typhi,Helicobacter,and Escherichia coli.It is proposed that the gallbladder may be the result of the joint action of inflammation with the bacterial flora.Similarly,the inflammatory “tumor infiltrating lymphocyte” (TIL)can be observed in the tumor and its surrounding tissues,and may also play a role in tumor growth and metastasis.However,detailed mechanisms about the relationship between inflammation and gallbladder cancer is still not clear.No specific anti-inflammatory drugs for gallbladder cancer have been developed. In the near future,anti inflammatory drugs may play a more important role in gallbladder cancer prevention and treatment.

Objective To investigate influence factors on neonatal necrotizing enterocolitis (NEC) and find a preventive way against the said disorder. Methods A retrospective study was conducted of 63 cases NEC (observation group) and 70 cases non-NEC (control group) admitted to the neonatal intensive care units (NICU) of two hospitals from January 2010 to May 2014. The cases were grouped in 29 factors. The individual factors of 28 out of the 29 factors were analy-sised with SAS 9.1 statistical software, and the screened important factors were investigated by logistic regression. Re-sults The ratios of 4 factors, application of prenatal corticosteroids, breast feeding prior to the ailment, oral probiotics and hyperbilirubinemia in the observation group were lower than those in the control group, while the ratios of 14 factors were higher than those in the control group, premature rupture of membrane, asphyxia, respiratory distress syndrome, respiratory failure, umbilical venous catheters, oxygen uptake, sepsis, septic shock, disseminated intravascular coagula-tion (DIC), congenital heart disease, scleredema, hyponatremia, hypocalcaemia and transfusion. The differences of fac-tors mentioned were of statistic significance (P<0.05), while the differences of other factors were not statistically signifi-cant between the two groups (P>0.05). Logistic regression analysis indicated that breast feeding, oral probiotics and hy-perbilirubinemia were protective factors against NEC; while sepsis, congenital heart disease and transfusion were major factors that lead to NEC. Conclusion NEC resulted from a combination of multiple factors, with sepsis, congenital heart disease and transfusion as the leading ones. Breast feeding, oral probiotics, and non-excessive curing against hyper-bilirubinemia will help reduce the incidence of NEC.

Objective:To investigate the curative effect of the Pavlik harness and polyester material casting bandage for the early treatment of congenital knee dislocation (CDK).Methods:The clinical data of 12 patients (13 knees) with CDK treated in Provincial Hospital Affiliated to Shandong First Medical University from December 2011 and January 2019 were studied retrospectively.There were 3 males and 9 females.The median age at the initial visit was 3 days ( 1-29 days). Eleven patients had unilateral CDK, and 1 patient had bilateral CDK.There were 5 cases(5 knees) with dislocation and 7 cases(8 knees) with subluxation.Associated musculoskeletal anomalies were observed in 7 patients, which included developmental dysplasia of the hip (DDH) in 6 patients, bilateral DDH and right clubfoot in 1 patient.The Pavlik harness was applied to keep the knee in position for 1.5-2.0 months if passive flexion achieved 90°or more.If passive flexion was below 90°, the polyester material casting bandage was employed to increase knee flexion, and the bandage was changed once a week.After passive flexion reached 90°, the patient was treated with the Pavlik harness for 1.5-2.0 months.Cases with DDH who worn the Pavlik harness were evaluated by ultrasonography in the follow-up period, and cases with clubfoot were treated with Ponseti polyester material casting bandages.Results:Three children (4 knees) were directly fixed with Pavlik harness for 1.5-2.0 months, 9 children (9 knees) were fixed with Pavlik harness for 1.5-2.0 months after 1-8 times (average 2.9 times) of plaster fixation. No plaster and Pavlik harness complications were found during the treatment.Twelve patients were followed up for 29.8 months on average (12.0 to 65.0 months). Ten patients (11 knees) were rated excellent, and 2 patients (2 knees) were rated good.Conclusions:The Pavlik harness and polyester material casting bandage for early treatment of CDK is simple, effective and safe.Associated musculoskeletal anomalies can also be corrected in the treatment process.The knee joint dislocation should be treated at first.

Objective: To understand the epidemiological characteristics of outbreaks and analyze the genetic characteristics of the whole genome of influenza H3N2 virus among avian-human-swine, and to elaborate the source of influenza virus. Methods: The epidemic information was collected using the case investigation, the pharyngeal swab samples from influenza-like-illness cases were detected by real-time PCR and virus isolation. The phylogeny and molecular features of whole-genome were analyzed with EditSeq and MEGA 5.05 software. Results: The prevalence rate of this outbreak was 34.88%, 15 samples of throat swabs were collected, the positive rate of nucleic acid detection was 73.33%, 5 strains of seasonal influenza A (H3N2) influenza viruses were isolated. The phylogenetic analysis showed the eight gene segments of the isolated influenza viruses belonged to the same cluster with 2015-2016 influenza vaccine strain A/Switzerland/9715293/2013(H3N2), and no recombination was found. Compared with vaccine strain, 14 variant amino acids of protein of HA were identified, and 8 of them were located in antigenic sites. All strains were sensitive to neuraminidase inhibitors while they showed resistance to blockers of M2 ion channel. The glycosylation sites analysis showed that two new glycosylation sites NRT151-153 and NAT245-247were added. Conclusions The outbreak was caused by seasonal influenza A (H3N2) virus which had an antigenic drift and no genetic avian-human-swine recombination was found.

Objective To investigate the correlation between lung Point-of-Care ultrasound score and clinical indicators by analyzing the features of Point-of-Care ultrasound in acute COVID-19.Methods 30 COVID-19 patients admitted to the Affili-ated Hospital of North Sichuan Medical College were selected,lung Point-of-Care ultrasound sequential scan was conducted in pa-tients with the BLUE-PLUS regimen and the lung Point-of-Care ultrasound semi-quantitative scoring was performed.The clinical indicators of partial pressure of carbon dioxide,anion gap,PH value,standard bicarbonate,oxygen partial pressure,oxygen satu-ration,the lymphocyte percentage(LYM% ),the lymphocytic absolute value(LYM#),the clinical typing,hospital stay,etc were collected.The lung Point-of-Care ultrasound score of acute COVID-19 patients in different clinical typing was compared and the features of Point-of-Care ultrasound in acute COVID-19 were analyzed.The ROC curve was constructed to analyze the effec-tiveness of lung Point-of-Care ultrasound score for the typing of normal type and(critical)severe type.Results In 30 cases of COVID-19 patients during the acute period,there were 29 patients with at least one lung point involvement on both sides(96.7% ),1 case with at least one lung point involvement on one side(3.3% )and 15 cases showed Lung consolidation＞1cm at least one lung point.Comparison of the same lung point between the left and right lung was performed and there was statistical-ly significant difference in the total scores between the left and right lung(P＜0.05).Comparison of total scores of different lung points in patients with different clinical types showed there was statistically significant difference in the total scores of left lung,the total scores of right lung,the total scores of whole lung between normal type and critical type(P＜0.05).The area under the curve(AUC)for the diagnosis of normal and(critical)severe types based on the total lung scores is 0.870.The total scores of the left lung,right lung,and whole lung were not correlated with LYM% ,LYM#,and anion gap(P＞0.05)were positively cor-related with clinical typing and hospital stay,and negatively correlated with standard bicarbonate(P＜0.05).The total scores of the left lung and whole lung were negatively correlated with PH value,oxygen partial pressure,and oxygen saturation,but posi-tively correlated with partial pressure of carbon dioxide(P＜0.05).The total scores of the right lung was not correlated with PH value,partial pressure of carbon dioxide,oxygen partial pressure,and oxygen saturation(P＞0.05).Conclusion There were differences in Point-of-Care ultrasound features among patients with different clinical types of acute COVID-19,Lung Point-of-Care ultrasound semi-quantitative scoring is correlated with pH value,partial pressure of carbon dioxide,standard bicarbonate,ox-ygen partial pressure,oxygen saturation,clinical typing and hospital stay.The patients of critical type have higher scores,lung Point-of-Care ultrasound is expected to be used for clinical typing and condition evaluation of acute COVID-19 patients.