Journal of Jiangsu University(Medicine Edition)

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Journal of Jiangsu University(Medicine Edition)

	2012 Vol.22 Issue.4 Published 2012-07-30

Article

	277	Exosome derived from human umbilical cord mesenchymal stem cell alleviates myocardial ischemia/reperfusion injury

		[Abstract]Objective: To investigate the repair effect of exosome derived from human umbilical cord mesenchymal stem cells(hucMSCexosome) to ischemia/reperfusion (I/R) myocardial cells. Methods: In vivo, Female SpragueDawley rats′ left anterior descending coronary artery (LAD) was occluded to induce regional myocardial ischemia, reperfusion was achieved by releasing the clamp. After 30 min of ischemia, hucMSCexosome was injected via tail vein. In vitro, I/R of H9C2(21) rat embryonic cardiac myoblasts was achieved by culturing the cells in low glucose DMEM without FBS in a hypoxia chamber, saturated with 5%CO2/93%N2 for 24 h and following reoxygenation using 10%FBS high glucose DMEM and hucMSCexosome in the normal incubating condition for 24 h. Using MTT, TUNEL, myocardial enzyme spectrum and mitochondrial membrane potential and other methods to detect the proliferation and apoptosis of myocardial cell. Results: In vivo,after the injury of I/R,lower level of LDH and CK activity was observed in the hucMSCexosome or hucMSC pretreated cells as compared with PBS control cells. TUNEL staining showed that there was a decrease in apoptosis in the injured cells after pretreated with hucMSCexosomes or hucMSC compared to PBStreated group. In vitro, cell proliferation examined by MTT assay were shown to reduce significantly after the injury of H/R. Pretreatment of cells with hucMSCexosome resulted in a marked decrease in hucMSCexosome induced mitochondrial membrane potential(ΔΨm) and apoptosis. Conclusion: Exosome secreted by hucMSC can reduce the injury of myocardial I/R effectively.
		2012 Vol. 22 (4): 277-281 [Abstract] ( 1763 ) [HTML 1KB] [ PDF 1250KB] ( 2169 )

	282	Expression,purification and identification of mIL-17RAaa32-322

		［Abstract］Objective: To clone the extracellular region of mouse interleukin17 receptor (mIL17RAaa32-322) and mouse IgG2AFc (mIgG2AFc), construct the recombinant prokaryotic expression vector of mIL17RA aa32-322mIgG2AFc, and then to express, purify and identify the fusion protein.Methods: The prokaryotic expression vector pET32a (+)mIL17RAaa32-322mIgG2AFc was constructed by using recombinant DNA technology and expressed in E. coli Rosetta. Then mIL17RAaa32-322mIgG2AFc fusion protein was purified by nickelchelating chromatography and identified by SDSPAGE electrophoresis and Western blot assay. Results: The prokaryotic expression vector pET32a(+)mIL17RAaa32-322mIgG2AFc was successfully constructed, which can express fusion protein of high purity. The mIL17RAaa32-322mIgG2AFc was confirmed by Western blot. Conclusion: Recombinant prokaryotic expression vector pET32a(+)mIL17RAaa32-322mIgG2AFc was successfully constructed and mIL17RAaa32-322mIgG2AFc fusion protein was purified for the research and application of foundation in the treatment of autoimmune disease.
		2012 Vol. 22 (4): 282-285 [Abstract] ( 1605 ) [HTML 1KB] [ PDF 899KB] ( 1994 )

	286	Effect of OsmY on gene expressional regulation of Salmonella enterica serovar Typhi at early stage of hyperosmotic stress

		Objective: To explore the effect of OsmY on gene expressional regulation of Salmonella entericaserovar Typhi (S.Typhi) at the early stage of hyperosmotic stress.Methods: The osmY gene deleted mutant of S.Typhi was generated through homologous recombination mediated by a suicide plasmid; the gene expression profiles of the wild type strain and the osmY deleted mutant at early stage of hyperosmotic stress was investigated by genomic microarray assay; qRTPCR was performed to validate the results of microarray assay. Results: The osmY deleted mutant of S.Typhi was prepared successfully; analysis of genomic microarray assay showed that 27 genes were up regulated and 128 genes were down regulated in the osmY deleted mutant at the early stage of hyperosmotic stress compared to the wild type strain. The results of qRTPCR assay were consistent with the results of microarray assay. Conclusion: osmY of S.Typhi may be a regulator played an important role in regulating gene expression at early stage of hyperosmotic stress.
		2012 Vol. 22 (4): 286-290 [Abstract] ( 1774 ) [HTML 1KB] [ PDF 866KB] ( 1998 )

	291	Methotrexate inhibited the development of collagen-induced arthritis in mice by decreasing the ratio of Th17/Treg cells

		Objective: To investigate the therapeutic effect of methotrexate (MTX) to mouse collageninduced arthritis (CIA) and its possible mechanism. Methods: MTX was injected into local joint of CIA mice three times a week from the 22nd day to the 50th day after primary immunization, and PBS was set up as negative control. The incidence and arthritis score in joint were calculated and compared between each group. The histopathological changes were evaluated in paraffin sections. The percent of Th17 cells and Treg cells in inguinal lymph nodes were analyzed by Flow cytometry (FCM). Results: MTX reduced the arthritis incidence and severity score of CIA mice effectively (P＜0.001). Paraffin sections showed that MTX inhibited the inflammatory cells infiltration, pannus formation, and destruction of cartilage and bone. MTX decreased the percent of Th17 cells and increased the percent of Treg cells effectively (P＜0.05). Conclusion: MTX inhibited the development of rheumatoid arthritis effectively. The mechanism may be involved in which the ratio of Th17/Treg cells was decreased.
		2012 Vol. 22 (4): 291-294 [Abstract] ( 1261 ) [HTML 1KB] [ PDF 1503KB] ( 2449 )

	295	*Compare the effect of resistance to polymyxin B between virK* and mig-14 gene of Salmonella enterica serovar Typhi**

		Objective: To compare the effect and study the association in resistance to polymyxin B between virK and mig-14 gene of Salmonella enterica serovar Typhi. Methods: Prepared the virK deleted mutant and virK/ mig-14 double deleted mutant of S. enterica serovar Typhi by homologous recombination mediated by suicide plasmid；performed the polymyxin B sensitivity assay for wild strain,mig-14 deleted mutant,virK deleted mutant,virK/ mig-14 double deleted mutant and phoP deleted mutant,then compared their resistance to polymyxin B. Results: Successfully prepared a virK mutant and virK/mig-14 double mutant. The effect of polymyxin B on mig-14 mutant,virK mutant and virK/mig-14 double mutant were nearly similar at poor acid environment from the polymyxin B sensitivity assay．Conclusion: VirK participated in the regulations of resistance to polymyxin B. The resistance to polymyxin B of virK mutant was nearly consistent with mig-14 mutant．
		2012 Vol. 22 (4): 295-298 [Abstract] ( 1007 ) [HTML 1KB] [ PDF 1054KB] ( 1927 )

	299	Establishment of the realtime fluorescence quantitative PCR assay for detection of murine adipose tissue chemokine CCL20 mRNA expression
		CHEN Yan-Hong, ZHU Chen-Lu, WEI Yan-Cai, QIN Wei, TIAN Jie, YUAN Jing, CHEN Juan, WU Jing-Jing, TANG Xin-Yi, XU Hua-Xi, WANG Sheng-Jun

		2012 Vol. 22 (4): 299-301 [Abstract] ( 1439 ) [HTML 1KB] [ PDF 864KB] ( 1934 )

	302	Tumor-associated macrophage impacts anti-tumor responses through activation of liver X receptors
		ZHONG Xiao-Jing-1, 2 , Wang-Xiu-Zhen-3, Sheng-Hong-Guang-2
		Objective:To investigate the connection between tumor microenvironment and liver X receptors(LXRs), and their function on the development of tumorassociated macrophage. Methods: Macrophages were sorted from tumor tissue and peritoneal lavage fluid, the expression level of LXRs, IL10 and IL12 were determined by realtime PCR. The internal relations among microenvironment , LXR receptors and TAM were explored in seperated experiments as follows: Macrophages cell line RAW264.7 were cocultured with tumor cell line TC1 ,or treated with tumor supernatant or LXR agonists , or the LXR signaling pathway in macrophages were blocked, then the phenotypes and functions of macrophages were determined. Results: Tumor microenvironment could promote the expression of LXR receptors, and induce the M2 subset phenotype of macrophages. The activation of LXR receptors could also induce the M2 phenotype, and the effect could be reversed when the LXR signaling pathway in macrophages were blocked. Conclusion: LXR could be activated by tumor microenvironment, then induce the accumulation of TAM, and activate their function on promote the growth, metastasis, and immune evasion of tumor.
		2012 Vol. 22 (4): 302-306 [Abstract] ( 1381 ) [HTML 1KB] [ PDF 1003KB] ( 2377 )

	307	Construction of SOCS3 3′-UTR luciferase reporter vector and identification of its activity
		LIU Xiao-Mei-1, Pang-Rong-Rong-2, Zhao-Dan-2, Li-Yan-2, Dan-Kai-2, Wang-Ying-Wei-2
		[Abstract]Objective: To study the microRNA(miRNA) targeting to 3′untranslated region(3′UTR) of suppressors of cytokine signaling3(SOCS3) gene, a SOCS3 3′UTR luciferase reporter vector was constructed and to analyse the miRNAs regulated SOCS3 expression through detecting the luciferase activity of SOCS3 3′UTR. Methods: The 3′UTR fragment of SOCS3 gene was amplified by PCR from genomic DNA of primary astrocytes of mouse and cloned into luciferase reporter vector (pGL3Promoter), then the recombinant pGL3Promoter/SOCS3 was identified. The miRNAs targeting SOCS3 3′UTR was predicted by Target Scan5.2, RNAhybrid and FINDTAR3 softwares. Thereafter, pGL3Promoter/SOCS3 and miRNAs were cotransfected into HEK 293T cells and the luciferase activity of SOCS3 3′UTR was measured. Results: 3′UTR fragment of SOCS3 gene was successfully cloned into the pGL3Promoter reporter vector, which verified by Xba I digestion and DNA sequencing. The predicted miRNAs targeting SOCS3 3′UTR included miR203, miR291a5p, miR9, miR140 and miR130b. Compared with the control group, the luciferase activity of pGL3Promoter/SOCS3 treated with miR203, miR291a5p, or miR140 was remarkably decreased, respectively. Conclusion: The SOCS3 3′UTR luciferase reporter vector was constructed successfully, and the luciferase activity of the recombinant vector can be suppressed significantly by miR20, miR291a5p or miR140.
		2012 Vol. 22 (4): 307-311 [Abstract] ( 2825 ) [HTML 1KB] [ PDF 1431KB] ( 3265 )

	312	Adaptive evolution analysis of hepatitis B virus epidemic strains in China
		HUANG Yan, Zhang-Chao, Liu-Jun, Xu-Qing-Gang
		Objective: To detect evolution of hepatitis B virus(HBV), its genotypes B and C circulating in China, and to evaluate the association of positive selection on four HBV gene regions \[S, C, X, and reverse transcriptase (RT)\] with immune escape. Methods: All available fulllength genomic sequences (884 sequences) of HBV genotypes B and C in China were downloaded from GenBank. Four gene segments of S, C, X, and RT were obtained from the genomic sequences according to HBV reference sequence. Positively selected sites occurring in HBV genome were detected by HyPhy method. Results: A total of 101 and 125 positively selected sites were identified in genotype B and C respectively. Interestingly, in genotype B,37 positively selected sites were located in 32 Tcell epitopes, and in genotype C, 48 positively selected sites were located in 43 Tcell epitopes, suggesting an association of positive selection with viral immune escape. In addition, three known drug resistance sites 180, 181, and 223 in HBV genotype B RT gene, and four known drug resistance sites 173, 180, 181, and 223 in genotype C RT gene were also identified under positive selection, suggesting an association of positive selection with drug resistance. Conclusion: Drug resistance mutation and immune escape of HBV might be one major factor driving the adaptive evolution of HBV.
		2012 Vol. 22 (4): 312-319 [Abstract] ( 1718 ) [HTML 1KB] [ PDF 1001KB] ( 2446 )

	320	Establishment of a rabbit model of pulmonary fibrosis induced by stomach lavaging of paraquat
		GUO Shu-Hua, YIN Jiang-Ning, REN Guo-Qing, CAI Hua-Zhong
		Objective: To develop a method of establishing rabbit model of paraquatinduced pulmonary fibrosis. Methods: Twentyfive rabbits were divided randomly into the experimental group (n=20) and control group(n=5)．Paraquat (40 mg/kg) was administrated to the animals of the experimental group by stomach lavaging once of paraquat. The rabbits were killed on the day 3，7，14 and 28 after paraquat administration．The rabbits in the control group were treated by stomach lavaging once of physiological saline and killed on the 28th day. The general conditions, macroscopic structure and pathological changes of lung in the rabbits of both groups were examined and analyzed. Results: The manifestation of intoxication was shown at 1 h after paraquat administration, and obvious fibrosis of the lung tissue was observed on the day 28 in the experimental group. Conclusion:A rabbit model of pulmonary fibrosis can be established reliably by stomach lavaging of paraquat．
		2012 Vol. 22 (4): 320-323 [Abstract] ( 1363 ) [HTML 1KB] [ PDF 2393KB] ( 1732 )

	324	Studiesof siRNA MTA1 on anoikis of human bladder cancer cell line BIU-87
		CUI Fei-Lun-1, 2 , Qiu-Zhen-1, Hu-Jian-Peng-1, Fan-Yu-2
		Objective: To observe the effects of MTA1 small interfering RNA（siRNA）on anchorage-independent growth and anoikis of bladder cancer cell line BIU-87. Methods: After construction of the MTA1 gene eukaryotic expression vector of siRNA and transfected to the bladder cancer cell line BIU-87, the expression of MTA1 gene was detected by real-time PCR and western blot; to detect anchorage-independent growth using clon formation in soft agar,and anoikis using DNA fragmentation assay and flow cytometry assay，respectively. Results: The level of mRNA and protein of MTA1 of groups transfectd with siRNA were decreased markedly in a time-and dose-dependent manner. MTA1 siRNA can inhibit anchorage-independent growth in a dose-dependent manner. MTA1 can reduced the resistance to anoikis of BIU-87 cells in a dose-and time-dependent manner. Conclusion: MTA1 siRNA can inhibit the growth of bladder cancer cell line BIU87 through inducing anoikis.
		2012 Vol. 22 (4): 324-327 [Abstract] ( 1589 ) [HTML 1KB] [ PDF 933KB] ( 1890 )

	328	Mechanism of miR let-7b inhibiting breast cancer MCF-7 cells migration
		QU Jie-1, WANG Sheng-Lin-2, 吕Xi-Ying-3 , LI Qing-Shan-3, YANG Zong-Wei-4
		Objective: To observe the effect of miR let-7b on inhibiting breast cancer MCF-7 cells migration and to investigate it′s mechanism. Methods: miR let-7b were composed and transfected into breast cancer MCF-7 cells. MiR-control was taken as a reference. The migration of the cells was tested by scratch test and the IL-6 level of MCF-7 cells before and after transfected was tested by PCR and west-blot. Then the combination of miR let-7b and IL-6 was verification by Bioinformatics software and Dual luciferase. Results: Compared with the control group,miR let-7b made the migrating ability of MCF-7 cells reduced,while the anti-miR let-7b strengthen it.Those breast cancer MCF-7 cells which were transfected by miR let-7b had a lower level of IL-6,while those transfected by anti-miR let-7b had a higher level of IL-6.It was verificated by Bioinformatics software and Dual luciferase that miR let-7b was combined with IL-6. Conclusion: miR let-7b inhibited the expression of IL-6 of breast cancer MCF-7 cells at the genetic level and then inhibited the migration.
		2012 Vol. 22 (4): 328-331 [Abstract] ( 2181 ) [HTML 1KB] [ PDF 918KB] ( 2179 )

	332	*Detection of biofilm formation and biofilm-associated genes of Staphylococcus aureus* clinical isolation**
		PAN Hong-1, Luo-Yu-Cheng-1, Zhao-Jian-Zhong-2, Duan-Xiu-Jie-2, Shao-Shi-He-1
		Objective: To detect biofilm formation and biofilmassociated genes of S.aureus clinical isolation. Methods: The biofilm formation was determined by semiquantitative microplate assay and scanning electron microscopy method, and both ica and sarA genes were detected by PCR in 92 strains of S.aureus clinical isolation. Results: All of 92 strains in this study could form biofilm, and carry the sarA gene; 78 strains (84.78%) carried the ica gene. Conclusion: The S.aureus clinical isolations had the ability of biofilm formation, and the gene of ica and sarA were prevalent in S.aureus.
		2012 Vol. 22 (4): 332-335 [Abstract] ( 1547 ) [HTML 1KB] [ PDF 1016KB] ( 2208 )

	336	Relationship between polymorphism sites of adiponection and it′s receptor gene and the susceptibility of colorectal cancer
		ZHANG Ying-1, 2 , FENG Quan-Lin-2, LIU Chang-Ming-2, DI Rong-Ke-1
		Objective: To explore the relationship between polymorphisms in adiponection and adiponection receptor gene and risk of colorectal cancer. Methods: A total of 370 patients with colorectal cancer and 370 healthy individuals were enrolled in the study, DNA was isolated from 3 ml whole blood obtained from each individuals, and the polymerase chain reaction and restriction fragment length polymorphism (PCRRFLP) was applied to detect the genotype of the polymorphisms in adiponection（rs266729, rs822395, rs822396, rs2241766, rs1501299）and adiponection receptor gene(rs12733285, rs1342387). The frequencies distribution of all genotypes in cancer and control group were compared. Results: There was significant difference of genotype distributions for the polymorphism site(rs12733285) in adiponection receptor gene in the colorectal cancer group（CC 91.89%, CT 8.11%, TT 0.0%）and control group（CC 86.49%, CT 13.51%, TT 0.0%）（χ2=5.61, P=0.018）; ② There was significant difference distribution of rs1342387 polymorphism in adiponection receptor gene genotypes in cancer group（GG 48.65%, AG 38.92%, AA 12.43%）and control group（GG 37.84%, AG 46.49%, AA 15.68%）（χ2=8.87, P=0.012）. ③ The CT genotype of rs12733285 was associated with decreased CRC risk(CT versus CC:adjusted OR=0.56, 95% CI=0.35-0.91), and the AG and AA genotypes of rs1342387 were associated with decreased CRC risk(AG versus GG: adjusted OR=0.66, 95% CI＝0.48-0.90; AA versus GG: adjusted OR=0.62, 95% CI＝0.39-0.98; AG/AA versus GG: adjusted OR=0.65, 95% CI＝0.48-0.87）. ④No significant difference of genotype frequencies distribution was observed for the polymorphism sites in adiponection gene between cancer group and control group. Conclusion: Polymorphism sites in adiponection receptor gene(rs12733285、rs1342387) were associated with colorectal cancer risk.
		2012 Vol. 22 (4): 336-341 [Abstract] ( 1708 ) [HTML 1KB] [ PDF 744KB] ( 2349 )

	342	Curative observation of Epi-LASIK and LASIK for high myopia
		WU Deng-Lei, ZHANG Qin, PAN Xiao-Ying
		Objective: To explore curative effect after microkeratome excimer laser epithelial keratomileusis (EpiLASIK) and laser in situ keratomileusis (LASIK) for high myopia, and to evaluate the safety and efficacy of EpiLASIK and LASIK. Methods: Used United States VISX StarS4 laser, and Moria flat push type micro corneal epithelial knife making corneal flap was used for EpiLASIK, with 0.02% mitomycin C sponge soaked in. LASIK used Moria rotating type corneal knife making 110 μm corneal flap. The patients was followed up more than 6 months,and compared postoperative symptoms, visual acuity, refraction, and so on. Results: One week postoperative,the proportion of UCVA≥0.8 of EpiLASIK (47.8%) was less than LASIK (95.8%), the difference was statistically significant (χ2=27.056, P= 0.000); no statistically significant differences were found in those of EpiLASIK(95.7%, 97.8%, 97.8%) and LASIK(97.9%, 97.9%, 97.9%) after 1,3,6 months. After 1 week, the diopter difference was statistically significant (t=-7.123,P= 0.000); after 1,3,6 months the diopter difference was not statistically significant. Conclusion: LASIK was still the mainstream of current treatment of myopia. EpiLASIK was an effective and safe procedure for high myopia. Although the recovery of visual acuity was slower than LASIK, it had a wide range of operation indication, its longterm efficacy still remains to be seen.
		2012 Vol. 22 (4): 342-345 [Abstract] ( 2472 ) [HTML 1KB] [ PDF 728KB] ( 1839 )

	346	Significance and expression of β-catenin and MMP-7 in gastric carcinoma
		LV Cheng-fang1, ZHAO Yuan2,3, WANG Yong-zhong2
		Objective: To study expression of β-catenin,MMP-7 and its significance in patients with gastric carcinoma. Methods: The expression of β-catenin in 75 tissue of gastric carcinoma was detected by immunohistochemistry ABC method, and the expriession of MMP-7 in the positive tissue of βcatenin in gastric carcinoma was detected by Western blot. Results: The protein of βcatenin was expressed in 35 of 75 cases in gastric carcinoma, the positive percentage is 46.67%. However, the protein of β-catenin was expressed in 3 of 30 cases in normal gastric tissue, the positive percentage is 10.00%. There was a significant difference in statistical analysis (P<0.05). In addition, The protein of β-catenin was expressed in 44 of 62 cases in patients with lympha node metastasis, the positive percentage was 70.97%. And the positive percentage of β-catenin was 46.15% in patients without lympha node metastasis (P<0.05). The positive rate for β-catenin and MMP-7 were 73.91%, 65.22% respectively in poor-differentiated gastric carcinoma, while they were 47.36%, 42.10% respectively in moderately differentiated gastric carcinoma and 50.00%, 40.00% respectively in well differentiated gastric carcinoma. There was a significant difference in statistical analysis (P<0.05). The positive immunostaining rate for MMP-7 in the area of a positive immunostaining reaction of β-catenin was high in gastric carcinoma (60.00%). The expression of MMP-7 was significantly correlated with β-catenin expression (r＝0.617,P<0.01).Conclusion: The expression of β-catenin, MMP-7 in gastric carcinoma are related to the infiltration and metastasis of gastric carcinoma.
		2012 Vol. 22 (4): 346-349 [Abstract] ( 1278 ) [HTML 1KB] [ PDF 800KB] ( 1807 )

	350	Expression and clinical significance of nuclear transcription factor NF-κB in colon cancer
		PU Yong， CHEN Suo-cheng
		Objective: To investigate the expression and clinical significance of nuclear factor(NF)-κB in colon cancer. Methods: NF-κB subunit p65 mRNA and protein from 108 cases of colon cancer tissue were detected by RTPCR and Western blot, relationships between them and demographic informations(gender, age, high-fiber diet, alcohol),clinic pathologic factors (tumor location, size, histological type, depth of invasion, differentiation, lymph node metastasis, Dukes points Period) were analyzed. Results: In colon cancer patients, expression of NF-κB subunit p65 mRNA and protein differed with different histological types of tumor, depth of invasion groups, whether lymph node metastasis or not at Dukes stage, in which, NF-κB mRNA and protein expression were significantly higher in the mucinous carcinoma (signet ring cell carcinoma, mucinous adenocarcinoma), invaded serosa, lymph node metastasis and Dukes C+D (P<0.01 or <0.05). However, NF-κB mRNA and protein expression showed no difference with gender, age, highfiber diet, alcohol, and tumor location, size and degree of differentiation (P>0.05). Conclusion: NF-κB expression had something to do with cancer invasion and metastasis; it showed potential value to detect NF-κB subunit p65 mRNA and protein in the diagnosis and treatment of colon cancer.
		2012 Vol. 22 (4): 350-353 [Abstract] ( 1555 ) [HTML 1KB] [ PDF 756KB] ( 1807 )

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		2012 Vol. 22 (4): 354-355 [Abstract] ( 1336 ) [HTML 1KB] [ PDF 733KB] ( 2046 )

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		2012 Vol. 22 (4): 356-358 [Abstract] ( 1108 ) [HTML 1KB] [ PDF 712KB] ( 1699 )

	359


		2012 Vol. 22 (4): 359-362 [Abstract] ( 910 ) [HTML 1KB] [ PDF 723KB] ( 1833 )

	363


		2012 Vol. 22 (4): 363-366 [Abstract] ( 916 ) [HTML 1KB] [ PDF 782KB] ( 2800 )

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		2012 Vol. 22 (4): 367-368 [Abstract] ( 689 ) [HTML 1KB] [ PDF 692KB] ( 1776 )

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