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Objective@#To evaluate the differential calretinin immunostaining in different segments of total colonic aganglionosis and its utility in the diagnosis.@*Methods@#Nine specimens including ileum and colon segments were obtained from 9 patients with total colonic aganglionosis (TCA), from 2010 to 2016 year, in Wuhan Children′s Hospital, Tongji Medical College, Huazhong University of Science and Technology. Another 9 ganglionic specimens including the same segments from patients with non-Hirschsprung disease (non-HD) patients were collected as control. All cases were immunostained with calretinin. The patterns of calretinin immunostaining were observed, and morphometric analysis of each sample was performed by image analysis program (Image-Pro-Plus). The mean absorbance was evaluated by calculating the areas of the lamina propria occupied by the positively stained area of the calretinin at high power field.@*Results@#The same pattern of calretinin immunostaining was seen in ganglionic ileum and ganglionic colon segments, with staining seen in intrinsic nerves fibers (INF), and in granular aggregates in the lamina propria and muscularis mucosae. There was no significant difference in the numbers of calretinin-positive INF from the ganglionic segments. In contrast, the number of calretinin-positive INF and granular aggregates in aganglionic segments were significantly lower than those in the ganglionic group (P<0.01). In the ileum transitional zone, scattered calretinin staining was observed, and the amount of calretinin-positive INF was significantly lower than those in the proximal segment of ganlionic ileum (P<0.01).@*Conclusions@#Since there is significant different expression of calretinin among the different segments from TCA, calretinin immunostaining has potential value in detecting TCA. It could be an important adjunctive method in detecting TCA in the future.
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Objective To investigate the role of APRIL in SW480 cell line.Methods The CRC model was established in the nude mice,all the mice were divided into 3 groups,the mice were separately treated with APRIL siRNA,pGC-vector and PBS solution.The APRIL mRNA was detected by RT-PCR and the APRIL protein was surveyed by the way of immunohis-tochemistry (IHC).The proteins of TIMP-3,Syndecan-1and MMP-9 also were assessed by IHC.Results ①Tumor mass in the group of nude mice injected with PBS (2.15±0.30 g)was significantly higher than the injection APRIL siRNA group (0.95±0.15 g,P0.05)compared with the injection of empty vector group (2.20±0.25 g).②APRIL mRNA/18S rRNA ratio (2.48±0.25)in the group of mice injected with PBS was signifi-cantly higher than the injection APRIL siRNA group (0.39±0.15,P0.05)compared with the injection of empty vector group (2.51±0.30).③SW480 cells injected with APRIL siRNA signifi-cantly inhibited invasion and metastasis.TIMP-3 Allred scores in three groups were 7.70±0.35,1.10±0.16 and 1.15± 0.12,Syndecan-1 protein was 7.80±0.30,1.05±0.20 and 1.10±0.22 MMP-9 protein was 1.20 ±0.10,8.00±0.25 and 8.20±0.20,respectively.Conlusion APRIL was closely connected with the growth and metabasis of CRC.
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Objective To prove the remarkable inhibitive effect of multiple siRNAs targeting a proliferation-inducing ligand (APRIL) on the human colorectal cancer cell.Methods We constructed a multiple short hairpin RNA(shRNA) expression vector containing four shRNAs (pG4) as well as four single one (pGsh644,pGsh1451,pGsh1938,pGsh2231) against APRIL gene in SW480 cell,and then transfected them into the human colorectal cancer cell line by cationic liposome.Ultimately,SW480 were screened by EGFP to obtain expression cell lines.APRIL expression levels including mRNA and APRIL protein were detected after transfected with all different kinds of vectors.Results A multiple shRNA expression vector containing four shRNAs (pG4) and four single ones were successfully constructed.Four single vectors (pGsh644,pGsh1451,pGsh1938,pGsh2231) and the multiple siRNAs expression vector (pG4) all decreased the APRIL mRNA by 56.2％,49.5％ ;50.9％,49.2％ and 79.3％.And APRIL protein expression was also remarkably reduced,especially by multiple siRNAs expression vector(87.5％).Conclusion Multiple siRNAs expression vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA.What we found suggested us using the vector containing multiple shRNA to silence the expression of APRIL might be exploited as a novel therapeutic strategy for tumors.
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Objective To construct and screen siRNA targeting a proliferation-inducing ligand (APRIL) gene in a mouse colorectal cancer celline, CT-26. To investigate the effects to the cell growth and migrant capacity of CT-26 after knockdown APRIL gene, lay the foundation for molecular targeted therapy to colorectal cancer. Methods Four pairs of APRIL siRNA were designed and chemically synthesized. And disorder sequences were synthesized as a negative control. These sequences were transfected with LipofectAMINE 2000 into CT-26 cells, which high-expressed APRIL gene. The transfection efficency rate of 6-FAM labelled control siRNA was detected by fluorescence microscope. The inhibition effectiveness of APRIL mRNA and protein was analyzed by FQ-RT-PCR and Western blot, respectively. Cell proliferation activity was analyzed by cell counting kit-8, cell migration capacity was detected by the repair of cell damage, and MMP-2 together with TIMP-1, two important regulatory genes in cell metastasis, were measured by RT-PCR.Results The different kinds of APRIL siRNA effectively suppressed the level of APRIL mRNA and the protein expression in CT-26 (P ＜ 0.05 ). Cell proliferation and metastasis ability were repressed after APRIL siRNA transfection( P ＜ 0.05 ), compared with random siRNA control and nontransfected control. The mRNA levels of MMP-2 and TIMP-1 genes wre significantly altered among APRIL siRNA groups and two control groups ( P ＜ 0.05). Conclusion We have constructed and screened a kind of siRNA (APsi737) targeting APRIL gene in a mouse colorectal cancer cell line, CT-26. APRIL siRNA can effectively inhibit the cell growth and migration capacity, maybe be regulated by MMP-2 and TIMP-1.
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Objective To investigate the effects of a proliferation-inducing ligand(APRIL)gene silencing by small interfering RNA(siRNA)on cell cycle and proliferation of colon carcinoma SW480 cells.Methods The siRNA plasmid vector targeting APRIL gene,named as siRNA-APRIL,was transfected into SW480 cells,transfected with scrambled vector as a nontargeting control and nontransfected group as another control.APRIL mRNA and protein expression were examined by real-time PCR and Western blot,respectively.Cell proliferation activity was analyzed by cell counting kit-8(CK-8),cell cycle was detected by flow cytometry,and p21 together with p27,two important regulatory genes in cell cycle,were measured by RTPCR.Results Compared with nontargeting control and nontransfected control,APRIL expression was inhibited significantly at both mRNA and protein level by siRNA-APRIL being transfected in SW480 cells(P ＜0.05).Cell proliferation ability was drastically repressed after siRNA-APRIL being transfected at 48 h,72 h and 96 h(P ＜ 0.05).After transfected 48 h,the percent of Go/G1 phase cell was significantly increased,S and G2/M phase cell were significantly decreased,the number of cell in apoptosis was increased and the expression of p21 and p27 mRNA were up-regulated(P ＜ 0.05).There was no significant difference when compared the two control groups each other(P ＞ 0.05).Conclusion siRNA-APRIL can effectively knockdown the expression of APRIL gene in SW480 cells,moreover,it can inhibit the cell proliferation and induce G0/G1 phase cell cycle arrest,which occurrence may involve in upregulation the mRNA expression of p21 and p27.
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RNA interference (RNAi) is a post-transcriptional suppression method of introduction of double-stranded RNA molecules by artificially induced. Recently RNAi has been widely used in xenografted tumors of nude mice in vivo. In order to inhibit the growth of tumor, some genes related with tumor such as anti -apoptosis gene, proliferation promoting gene, metastasis-associated gene are depressed via RNAi. Additionally, in the nude mice test, RNAi is utilized as assisting methed of radiotherapy and chemotherapy of human tumors.
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OBJECTIVE:To establish the quality standard of Welie capsule METHO DS :Fritillary bulb,rhizoma corydalis and magnolia bark in the capsule were ident ified with TLC;the contents of berberine hydrochloride was determined with TLC scanning RESULTS:Fritillary bulb,magnolia bark and rhizoma corydalis could be tested out in TLC chromatogram;berberine hydrochloride appeared good linear re lationship in the range of 0 1～1 0?g,r=0 9 991;and the average recovery w as 97 84%,RSD=2 86%(n=5) CONCLUSION:This method can be used to control the quality of Weile capsule