OBJECTIVE To analyze the reasons of ethylene oxide disinfection failure and give the countermeasures to guarantee the sterile quality.METHODS The investigation was carried out from the basic component of the whole disinfection,tracing and finding out the reasons of failure,and raising countermeasures.RESULTS Without meeting the sterile standards of the packages,incorrect packing method,without seriously checking and too much loading were the main failure reasons.CONCLUSIONS More education for the staff,good packing and education with the procedures of ethylene oxide disinfection are the key points to guarantee the sterile quality.

Objective To investigate the real-time regulatory effects of IFN-γ, programed death ligand 2(PDL2) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway on the adherence, proliferation and migration of human placenta-derived mesenchymal stem cells(hPMSCs) based on a finding that IFN-γ could enhance the expression of PDL2 in hPMSCs through JAK/STAT signaling pathway.Methods hPMSCs were isolated by using enzyme digestion method and then co-cultured with IFN-γ, anti-PDL2 monoclonal antibody (anti-PDL2 McAb) and JAK inhibitor, respectively.Real-time cell analysis (RTCA) was used to detect the dynamic changes in the adherence, proliferation and migration of hPMSCs following various interventions.Results IFN-γ remarkably suppressed hPMSCs proliferation during the period from 40 hours to 80 hours after intervention and also inhibited the non-targeted migration of hPMSCs.However, hPMSCs adherence was not affected by IFN-γ.Co-culturing hPMSCs with anti-PDL2 McAb significantly enhanced hPMSCs adhesion and inhibited their non-targeted migration, but had no significant effect on hPMSCs proliferation.Furthermore, the proliferation of hPMSCs co-cultured with IFN-γ and anti-PDL2 McAb was significantly inhibited as compared with that of anti-PDL2McAb treatment group.The adhesion, migration and proliferation of hPMSCs were significantly inhibited after co-culturing them with JAK inhibitor.Conclusion IFN-γ can remarkably suppress the proliferation and migration of hPMSCs.PDL2 can enhance the migration and inhibit the adhesion of hPMSCs.JAK/STAT signaling pathway is involved in regulating the adhesion, migration and proliferation of hPMSCs.

The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl-isomerase PIN1 gene, the mutation in exon 3 of beta-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of beta-catenin gene and differential expression of beta-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (chi2=32.63, P<0.05), especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; (2) 58.6% (17/29) HCC tissues showed beta-catenin protein accumulation in cytoplasm and nucleus. 46.2% (6/13) HCC tissues indicated beta-catenin protein accumulation with higher level of PIN1 expression, while 53.8% (7/13) HCC tissues indicated beta-catenin protein accumulation with lower level or trace of PIN1 expression (chi2=0.00, P>0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of beta-catenin, and accompanied by beta-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of beta-catenin gene in tissues with high PIN1 expression level (chi2=58.12, P<0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from beta-catenin gene mutation.

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.

Objective The purpose of this study is to observe the changes of immune cell subsets in lupus mice after umbilical cord mesenchymal stem cells (UC-MSCs) transplantation. Methods B6.MRL-Faslpr lupus mice were randomly divided into the following three groups: the UC-MSCs treated group, the fibroblast like synoviocytes (FLS) treated group and the untreated group. MSC (1×106) or FLS (1×106) were injected into the tail vein of lupus mice respectively. Four weeks after treatment, the spleen index was calculated. The pathological changes of kidney were assessed by HE staining. The frequencies of immune cell subsets in spleen and macrophage in kidney as well as abdominal cavity were analyzed by flow cytometry. Data were analyzed with t test. Results The spleen index of UC-MSCs treated lupus mice [(79 ±9) mg/10 g] and IgG level [(7.5±1.5) mg/ml] were significantly decreased when compared with FLS treated group [(147±23) mg/10 g, t=2.78, P<0.01] [(17.0 ±2.8) mg/ml, t=3.00, P<0.01] and the untreated group [(156 ±16) mg/10 g, t=4.29, P<0.01] [(16.7 ±1.6) mg/ml,t=4.01, P<0.01]. HE staining also showed that the pathological changes of kidney were alleviated after MSCs transplantation. In addition, the frequency of plasma cells in the untreated group [(2.61 2± 0.318)% vs (0.306±0.017)%, t=7.22, P<0.01] and the FLS treated group [(2.412±0.297)% vs (0.306±0.017)%, t=7.07, P<0.01] were markedly higher than MSCs treatment [(0.306 ±0.017)%]. Moreover, the frequency of CD25+Foxp3+/CD4+Treg in the MSCs treated group [(15.08±0.81)%] was significantly increased compared with the untreated group [(8.02 ±0.47)%, t=7.45, P<0.01] and FLS treated group [(8.80 ±0.23)%, t=7.39, P<0.01]. MSCs treatment resulted in a decrease in CXCR5+PD1+/CD4+Tfh and IFNγ+/CD4+Th1 subset, compared with the untreated group [(14.3±1.5)%vs (31.5±3.3)%, t=5.25, P<0.01] [(1.78±0.27)% vs (5.93±1.56)%, t=2.60, P<0.05] and the FLS treated group [(14.3±1.5)%vs (28.8±2.2)%, t=5.49, P<0.01] [(1.78±0.27)%vs (4.88±0.81)%, t=3.61, P<0.01]. The frequency of macrophage in kidney of the MSCs treated group [(3.52 ±0.37)%] was markedly increased compared with the untreated group[(1.58±0.29)%, t=3.25, P<0.01], while neither the IL4+/CD4+Th2 subset nor the IL17+/CD4+Th17subset and the frequency of macrophage in abdominal cavity showed significant changes in the three groups. Conclusion These findings suggest that the therapeutic effects of MSCs on lupus mice may mediate through increasing the frequency of spleen Treg and renal macrophage and decreasing the frequency of Tfh, Th1 and plasma cells.

Objective:To determine the difference of miRNA expression profiles in 6 types of human cancer cells by microarray technique.Methods:The microarray was prepared,with contained 210 oligonucleotides,including 206 probes complementary with human and mouse miRNA sequences and 4 positive control oligos.MiRNAs were extracted from HeLa(human cervical cancer epithelial cells),MCF-7(human breast cancer cells),A549(human lung adenocarcinoma cells),HT-29(human colonic cancer cells),ES-2(ovarian carcinoma cells),and K562(chronic myelogenous leukemia cells) cells and were labeled with Cy3 for hybridization to the miRNA microarray.The slides were scanned by ScanArrayTMExpress1.0 and images were analyzed by ScanArray3.0 and Cluster3.0;the results were confirmed by Northern blotting and RT-PCR.Results:Totally 115 miRNAs were found to be differentially expressed in the 6 cancer cell lines,with miR-21 expression up-regulated and miR-125b,let-7 expression down-regulated.The expression of miR-17-5p and miR-20a was in cluster and was more higher in ES-2 cells than in other cells.HeLa and MCF-7 cells were located on a single branch of the dendrogram in cluster analysis.Northern blotting showed that both pre-and miR-17-5p expressed in K562,pre-miR-17-5p was weakly expressed in A549 and ES-2 cells,and obvious pre-miRNA expression and weak miRNA expression were noticed in MCF-7,HeLa,and HT-29 cells.RT-PCR showed that expression of pre-miR-17-5p in K562 cells was higher than those in other cells.Expression of miR-21was high in all 6 cell lines and the highest expression was seen in A549 cells.Conclusion:Microarray can be used to detect miRNA expression files in cancer cells,which contributes to the study of the relation between miRNA and tumor.

This study explored the possibility that the components in melanoma cytoplasm induce murine BMSCs transformation and expression of Melan-A by morphologically observing the changes of BMSCs and immunocytochemically detecting Melan-A in the cells after culturing BMSCs in medium containing melanoma cytoplasm components (MCC). MCC of B16 melanoma cells was prepared and BMSCs were cultured and induced by adding the MCC into culture medium. The cells were morphologically observed and Melan-A was immunohistochemically detected to confirm BMSCs transformation. MCC-induced BMSCs underwent morphological changes. A number of melanin granules appeared in the cytoplasm of the cells and some were released into surrounding areas. Several cells that might come from one cell formed a cluster, and their granules, together with those secreted by other induced BMSCs, formed a so-called "sphere-formed structure". The induced BMSCs expressed Melan-A. We are led to conclude that there might be some factors in the cytoplasm of melanoma cells that might induce BMSCs transformation toward melanogenic cell, or even melanoma.

The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl- isomerase PIN1 gene, the mutation in exon 3 of β-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of β-catenin gene and differential expression of β-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (x2 =32.63, P＜0.05), especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; (2) 58.6% (17/29) HCC tissues showed β-catenin protein accumulation in cytoplasm and nucleus. 46.2% (6/13) HCC tissues indicated β-catenin protein accumulation with higher level of PIN1 expression, while 53.8% (7/13) HCC tissues indicated β-catenin protein accumulation with lower level or trace of PIN1 expression (x2 =0.00, P＞0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of β-catenin, and accompanied by β-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of β-catenin gene in tissues with high PIN1 expression level (x2=58.12, P＜0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from β- catenin gene mutation.

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS)stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-C1 in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasmid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) %and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.

Objective:To explore a method to obtain more mouse bone marrow cells more quickly.Methods:Take both lower limbs of the same mouse,the bone marrow cells were obtained by either the tube-nesting EP method or the traditional syringe irrigation method,recorded the time and quantity of bone marrow cells,to analyze the proportion of viable cells by flow cytometry,and to detect the proliferation of these bone marrow cells used CCK-8 method.Results:Compared with the traditional syringe irrigation method,the time of obtaining mouse bone marrow cells by tube-nesting EP method was shorter(P<0.000 1).Compared with the traditional syringe irrigation method,the number of bone marrow cells obtained by tube-nesting EP method was more(P<0.05).The proportion of live cells and proliferation ability of bone marrow cells obtained by the two methods were identical.Conclusion:Tube-nesting EP method is a new faster method to get more mouse bone marrow cells.