ABSTRACT
AIM: To investigate the effects of adenovirus-mediated human tissue kallikerin (Ad-hKLK1) gene delivery on the proliferation, migration of VSMC_(SHR) induced by platelet derived growth factor-BB (PDGF-BB). METHODS: The VSMC_(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin (MTT). The migration was assessed by modified Boyden chamber assay. Western blotting was used to determine the expressions of the cycle-independent kinase inhibitors p27~(Kip1) and p21~(Cip1).RESULTS: Proliferation of VSMC_(SHR) induced by PDGF-BB was inhibited after transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 fell on 100 MOI, with the peak inhibition rate of 39.3% (cell counting, n=3, P<0.01), 30.2% (MTT, n=3, P<0.01), peak stunning rate of cell-cycle in phase G0/G1 at 36.4%. The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery were significantly abolished by Hoe140, a bradykinin B2 receptor antagonist. Migration of VSMC_(SHR) induced by PDGF-BB was inhibited after hKLK1 gene delivery, with the peak inhibitory rate of 34.6% (n=6, P<0.01). However the inhibitory effects of migration were not blocked by Hoe140. The protein expression of p27~(Kip1) and p21~(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n=3, P<0.01, respectively).CONCLUSION: The hKLK1 gene delivery may inhibit the proliferation and migration of VSMC_(SHR) induced by PDGF-BB. Bradykinin B2 receptor probably mediates the up-regulating expression of p27~(Kip1) and p21~(Cip1) that contributes to the inhibitory effects of proliferation of hKLK1. However, the inhibitory effects of migration by hKLK1 gene delivery may not be mediated by bradykinin B2 receptor.
ABSTRACT
To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT) primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI377 analyzer. Then the KK gene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy.. .
ABSTRACT
To clone and sequence the human tissue kallikrein gene of Chinese, and to construct eukaryotic expression recombinant of KK, total RNA was extracted from human pancreas and human tissue kallikrein gene cDNA was amplified by PCR after reverse-transcription by using Oligo(dT)primer. The original kallikrein cDNA was recovered and filled with Klenow enzyme and inserted into KS plasmid. After restriction endonuclease digestion, KK cDNA was sequenced by ABI 377 analyzer.Then the KKgene was amplified from pBluescript KSKK and inserted into pcDNA3. A sequence comparison showed that the cloned kallikrein gene was only one nucleotide different from that reported in the Genbank. The coding amino acid was Asp in the Genbank gene, while the coding amino acid of Chinese kallikrein gene was Asn. The KK cDNA fragment was inserted into the eukaryotic expression vector pcDNA3. The cloned kallikrein gene and the pcDNA3KK can be used for further study in gene therapy...
ABSTRACT
Objective To investigate the distribution of human papillomavirus(HPV) subtypes in condyloma acuminatum(CA) patients in Shenzhen People's Hospital and assess the association between HPV subtypes and cervix neoplasm.Methods Type specific prevalence and extent of multiple infection were assessed in the genital tract CA samples collected from 352 patients visiting Departments of dermatology or gynecology of Shenzhen People's Hospital during 2003-2004.PCR using MY09/11 as primer and reverse dot blot hybridization for the genotyping of 9-20 HPV subtypes.Results HPV type diversity was broad in the investigated CA patients.The low risk HPV type(HPV subtype 11,6) was dominant and multiple HPV infections occurred in 37% of HPV-positive samples.High risk HPV type was dominant in CA from cervix,especially in the high grade cervical intraepithelial neoplasia(CIN Ⅱ+),and multiple high risk HPV(HR-HPV) infection was found in 87% of HPV positive samples.Conclusion HPV subtypes 6 and 11 were dominant in the patients visiting Shenzhen People's Hospital.HPV subtypes 16 and 18 may be the main causes of malignant changes of cervix.
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Objective: To study the effect on cytotoxicity and drug resistance by blocking ERCCl gene expression in o-varian cancer cell lines. Methods: ERGC1 antisense expression vector was constructed and transfected into human ovarian cancer cell lines. Northern blotting and Western blotting were used to detect the RNA and protein expression level in the cells. Luciferase assay system was used to reveal the host cell reactivation function. MTT assay was used to study the effect on cytotoxicity and drug resistance in the cell lines. Results: After transfection of the antisense ERCCl, Northern and Western blotting indicated that the RNA and protein expression level of ERCCl was obviously decreased. Luciferease assay showed reduced DNA-damage repair capacity as assessed by host cell reactivation. MTT assay also showed decreased drug resistance to cisplatin in the lines. Conclusion: Transfection of antisense ERCCl may enhance the cisplatin cytotoxicity by disturbing the NER pathway in cisplatin-resistant cell lines.
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The changes ot free radicals matebolism in liver of rat fed grains from a Keshan disease endemic area and the effects of selenium and vitamin E on those were studied. The endemic grains that were deficient in selenium caused markedly decrease in the glutathione peroxidase activity, and marked increase in the lipid peroxides content and the free radicals level of the rat livers. The diets that supplemented the endemic grains with either selenium (0.22 mg/kg diets) or DL-alpha-tocopherol (100 mg /kg diets) caused the falls in the lipid peroxides content and the free rdicaals level. There was a marked rise in the al pha-tocopherol content and reduced glutathione content in livers of rats fed grains from the endemic area, suggesting an increased reqiu-rement of vitamin E and production of glutathione in the selenium-defi-cient rats. The results indicate that a disorder of free radicals metaboli- sm is induced by the pathogenic factors existing in the grains cultiva-ted in a Keshan disease endemic area, and is reduced by supplementing the grains with sodium selenium or vitamin E, suggesting that relative lack of vitamin E may play an important role in the mechanism of Keshan disease onset.