ABSTRACT
@#【Objective】To explore the role of lncRNA Xist in proliferation and migration of rat bone marrow mesenchymal stem cells（BMSC）and its possible mechanism.【Methods】BMSC were isolated，cultured and identified from the femur and tibia of 3 weeks old SD female rats in vitro. SiRNAs was designed and screened to acquire a high silencing efficiency siRNA. Lipo2000 was used to transfected si- Xist and si- NC into BMSC of the experimental group（si- Xist group）and the control group（si-NC group）. BMSC proliferation capacity was determined by CCK-8 assay. The transverse and longitudinal mobility of BMSC were measured by wound healing assay and transwell migration assays. QPCR was performed to verify the silencing efficiency of lncRNA Xist and detect the expression levels of SDF- 1 and CXCR4 mRNA. Western blot was used to quantify the expression of CXCR4 protein.【Results】The P3 generation BMSC shows shuttle- like or whirlpool-like，and flow cytometry showed CD11b（-），CD34（-），CD45（-），CD44（+），CD90（+），CD105（+）. When siRNAs were used to interfere with the expression of lncRNA Xist in BMSC ，the silencing efficiency of three siRNAs was 67.92% ，68.72% and 98.32% ，respectively. CCK- 8 assay showed that the OD450 value of si- Xist group decreased compared with si-NC group at 24 h and 48 h（P < 0.001，P < 0.01，respectively）and had no statistical difference at 12 h（P > 0.05）. Wound healing assay showed that the wound healing percentage of si-Xist group was lower than that of si-NC group（P < 0.05）；and the transwell migration assay showed that，compared with si- NC group，the cells that migrated through the polycarbonate membrane were obviously decreased at 6 h（P < 0.001）. QPCR experiment showed that CXCR4 expression in si-Xist group was lower than that in si-NC group at mRNA level（P < 0.05），while SDF-1 expression showed no significant statistical difference（P > 0.05）. Western blotting confirmed that CXCR4 expression in si- Xist group was lower than that in si-NC group（P < 0.05）.【Conclusions】LncRNA Xist promotes proliferation and migration of rat BMSC by regulating CXCR4 expression.
ABSTRACT
Objective To analyze the mutation of single nucleotide polymorphism (SNP) loci in genes most significantly associated with acute rejection after liver transplantation by high-throughput sequencing. Methods Peripheral blood samples were collected from 68 recipients undergoing allogeneic orthotopic liver transplantation. According to the incidence of acute rejection, all patients were divided into the acute rejection group (n=13) and non-rejection group (n=55). Through the literature review, 44 mutant SNP loci associated with acute rejection were finally identified. Using 44 SNP loci as the detection targets, high-throughput sequencing analysis was performed to detect peripheral blood samples in two groups of recipients. Bioinformatics analysis revealed the mutation rate of the SNP loci of genes related to acute rejection. Results The mutation rate of SNP loci of the interleukin(IL)-10 TT genotype,T allele,AA genotype and A allele in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of the cell chemokine receptor(CCR)5 AG genotype in the acute rejection group was significantly lower than that in the non-rejection group,the mutation rate of the SNP loci of the CCR5 GG genotype in the acute rejection group was significantly higher than that in the non-rejection group.The mutation rate of the SNP loci of IL-4 CT genotype in the acute rejection group was significantly higher than that in the non-rejection group,the mutation rate of the SNP loci of IL-4 TT genotype in the acute rejection group was significantly lower than that in the non-rejection group. The mutation rate of the SNP loci of nuclear factor-kappa B inhibitor alpha(NF-κBIA)C allele in the acute rejection group was significantly higher than that in the non-rejection group.The mutation rate of the SNP loci of vitamin D receptor(VDR)CC genotype and C allele in the acute rejection group was significantly lower than that in the non-rejection group. Differences were statistically significant (all P<0.05). Conclusions High-throughput sequencing analysis shows the genes associated with acute rejection after liver transplantation.Among them,the mutation rate of the SNP loci is relatively high in IL-10 TT genotype,T allele, AA genotype,A allele,CCR5 GG genotype,AG genotype,IL-4 CT genotype,TT genotype,NF-κBIA C allele,VDR CC genotype and C allele.