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OBJECTIVE：To study the reversal effect of quercetin on human cervical squamous carcinoma cisplatin-resistant cell line SiHa/DDP. METHODS ：The drug resistance index of cisplatin to SiHa/DDP cells ，and the reversal resistance multiple of quercetin to SiHa/DDP cells were determined. The effects of quercetin （0.005 μg/mL），cisplatin（2.5 μg/mL），cisplatin combined with quercetin （2.5 μg/mL cisplatin+0.005 μg/mL quercetin），quercetin combined with pathway inhibitor（0.005 μg/mL quercetin+ 20 nmol/L rapamycin ）on the apoptotic rate of SiHa/DDP cells were investigated ，as well as its effects on the expression of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian rapamycin target protein （mTOR） signaling pathway related proteins （PI3K，Akt，mTOR，P-gp，p70S6K）. RESULTS ：The resistance index of cisplatin to SiHa/DDP cells was 5.19， and reversal resistance multiple of quercetin to SiHa/DDP cells was 4.00. Compared with cisplatin alone and quercetin alone ， cisplatin combined with quercetin ，quercetin combined with rapamycin could significantly increase the apoptotic rate of SiHa/DDP cells（P＜0.05），while decreased the phosphorylation of Akt ，mTOR and p 70S6K protein as well as the expression of P-gp protein （P＜0.05）. CONCLUSIONS ：Quercetin can effectively reverse drug resistance of SiHa/DDP cells to cisplatin ，which may be associated with inhibiting the expression of the protein related to PI 3K/Akt/mTOR signaling pathway.
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Objective To explore the expressions of clusterin and E-cadherin in cervical squamous tissues and their correlation.Methods Immunohistochemical technique was used to measure the expressions of clusterin and E-cadherin in 18 normal cervical tissues,32 cervical intraepithelial neoplasia (CIN)tissues and 40 cervical squamous carcinoma (CSC)tissues.Results There were on significant differences in baseline characteristics among the three groups based on standardized differences.The expressions of clusterin in CSC,CIN2 and CIN3 tissues were significantly higher than those in normal cervical tissues (P <0.05 ).The expression of clusterin was significantly lower in grade Ⅰ than in grade Ⅲ (P <0.01)and was connected with clinical stage (P <0.05 )instead of lymph node metastasis and cancer stromal invasion depth (P > 0.05 ).The expressions of E-cadherin in tissues of CSC, CIN2 and CIN3 were significantly lower than those in normal cervical tissues (P <0.05).E-cadherin expression was both significantly higher in grade Ⅰ and grade Ⅱ than in grade Ⅲ (P <0.05),and was associated with lymph node metastasis (P < 0.05 )rather than clinical stage and cancer stromal invasion depth (P > 0.05 ).A negative correlation was found between the expressions of clusterin and E-cadherin in CSC (r = - 0.339,P < 0.05 ). Conclusion The significantly different expressions of clusterin and E-cadherin may be related to the development of CSC,suggesting that the two may be used as potential markers for early diagnosis of CSC.
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Objective To explore the expressions of clusterin and E-cadherin in cervical squamous tissues and their correlation.Methods Immunohistochemical technique was used to measure the expressions of clusterin and E-cadherin in 18 normal cervical tissues,32 cervical intraepithelial neoplasia (CIN)tissues and 40 cervical squamous carcinoma (CSC)tissues.Results There were on significant differences in baseline characteristics among the three groups based on standardized differences.The expressions of clusterin in CSC,CIN2 and CIN3 tissues were significantly higher than those in normal cervical tissues (P <0.05 ).The expression of clusterin was significantly lower in grade Ⅰ than in grade Ⅲ (P <0.01)and was connected with clinical stage (P <0.05 )instead of lymph node metastasis and cancer stromal invasion depth (P > 0.05 ).The expressions of E-cadherin in tissues of CSC, CIN2 and CIN3 were significantly lower than those in normal cervical tissues (P <0.05).E-cadherin expression was both significantly higher in grade Ⅰ and grade Ⅱ than in grade Ⅲ (P <0.05),and was associated with lymph node metastasis (P < 0.05 )rather than clinical stage and cancer stromal invasion depth (P > 0.05 ).A negative correlation was found between the expressions of clusterin and E-cadherin in CSC (r = - 0.339,P < 0.05 ). Conclusion The significantly different expressions of clusterin and E-cadherin may be related to the development of CSC,suggesting that the two may be used as potential markers for early diagnosis of CSC.
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Objective To investigate the expression and significance of γH2AX in cervical squamous carcinoma.Methods Firstly,DNA were extracted from 74 cervical squamous carcinoma samples and PCR were tested for HPV infection.Secondly,formalin-fixed paraffin-embedded tissue sections (4 μm)were stained with H&E method to detect cervical lesions grading.Thirdly,HPV16 DNA were examined by in situ hybridization(ISH) and γH2AX,p16 were examined by immunohistochemical (IHC) staining.Then,30 cases typical tissue sections in which including the normal cervical tissue,cervical intraepithelial neoplasia and cervical carcinoma in situ were selected for comparing the HPV DNA loading,and the γH2AX and,pl6 expression.Finally,the feasibility of γH2AX serving as a biomarks in HPV infection-related cervical carcinogenesis were analyzed.Results In this study,HPV infection ratio is 98.65％,and HPV16 is the most common type with 74.32％ infection.In situ hybridization showed no HPV16 DNA exist in normal cervical tissues and CINI.In CIN Ⅱ HPV DNA exist mainly as episomal DNA.With the increasing of cervical lesions grade,HPV DNA was integrated into chromosome steadily.The expression of γH2AX and pl6 were positively associated with grading of cervical lesions.HPV DNA and γH2AX protein co-exist primarily in the prickle cell layer and the granular cell layer.The HPV DNA and p16 protein exist in different cell layer.Conclusion γH2AX may be employed as a biomarker for HPV positive cervical carcinogenesis.
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On integration into the host cervical keratinocyte genome, human papillomavirus (HPV) E7 protein binds pRB, releasing E2F from normally incompetent pRB-E2F complexes and allowing propagation of G1-S transition by the E2F. p16 INK4a, a tumour suppressor protein, increases in refl ex response to counter this. 29 histologically re-confi rmed low-grade squamous intraepithelial lesions (LSIL), 27 high-grade squamous intraepithelial lesions (HSIL) and 30 invasive cervical squamous carcinoma (SCC) were immunohistochemically stained for p16INK4a expression using the CINtec Histology Kit (REF 9511, mtm laboratories AG, Heidelberg, Germany) to re-affi rm the notion that integration of HPV occurs predominantly in SCC and possibly HSIL and less in LSIL and normal squamous epithelium (NSqE). Implicit was also the attempt to understand the role of E2F, as indicated by p16INK4a, in evolution of SCC from HSIL. No ethnic predilection was noted for LSIL, HSIL or SCC. Patients with SCC were signifi cantly older by about 14-years compared with HSIL (p<0.05) while there was no signifi cant age difference between HSIL and LSIL. p16INK4a expression was signifi cantly increased (p<0.05) in both HSIL (88.9%) and SCC (83.3%) compared with LSIL (3.4%) and NSqE (0%); the NSqE being normal squamous epithelium noted in 17 of the LSIL, 19 HSIL and 5 SCC. From these fi ndings there is suggestion that fundamental upstream events viz HPV integration, E7 upregulation followed by E2F activation occurs at point of transformation to HSIL and continues unrelentingly for another one to two decades before hitherto unclear factors convert a non-invasive lesion into an overtly invasive malignant counterpart. Interestingly, the occurrence of HSIL and LSIL in almost the same age group could mean that alteration from episomal to integrated form of HPV may not incur a prolonged incubation period, unlike from HSIL to SCC
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Objective: To investigate the role of indoleamine 2, 3-dioxygenase in the development of uterine cervical squamous carcinoma. Methods: From January 2008 to December 2008, 116 uterine cervical carcinoma specimens and 18 metastatic lymph node specimens from patients with CIN Ⅰ-Ⅲ and uterine cervical squamous carcinoma were evaluated for iDO expression by immunohistochemistry. Twenty normal cervical specimens and 20 normal lymph node specimens were used as the controls. Results: The expression of IDO was not found in normal cervix and CIN Ⅰ. In CIN Ⅱ, IDO expres-sion was weakly positive in 2 cases (2/10, 20%) and negative in 8 cases (8/10, 80%). In CIN Ⅲ, IDO expression was weak-ly positive in 8 cases (8/13, 61.5%), positive in 1 case (1/13, 7.7%) and negative in 4 cases (4/13, 30.8%). The positive ex-pression rate of IDO in cervical cancer stage Ⅰ -Ⅳ was 100% (83/83). In cervical cancer stage Ⅰ A and Ⅰ B, the positive ex-pression rate of IDO was significantly higher than that in CIN Ⅱ and CIN Ⅲ (P<0.01). The positive expression rate of IDO in cervical cancer stage Ⅱ A-Ⅳ B was significantly higher than that in Ⅰ A and Ⅰ B. IDO expression was associated with cervi-cal cancer progression (OR=0.807, P<0.01). IDO expression in primary lesions with lymph node metastasis was significant-ly higher than that in those without lymph node metastasis. IDO expression rate was 100% in metastatic lymph nodes. The IDO expression was not associated with cervical squamous carcinoma differentiation degree (OR=-0.139,P>0.05). Conclu-sion: In CIN Ⅱ, escape mechanisms that stimulate cervical squamous carcinoma progression is gradually developed. IDO expression in metastatic lymph nodes is possibly associated with immune tolerance. IDO expression is not associated with differentiation degree of cervical squamous carcinoma. IDO may be a prognostic factor for uterine cervical squamous carci-noma and a therapeutic target for treatment.
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Objective To investigate the infection of human papilloma virus(HPV) and expression of serum squamous cell antigen (SCCAg) in the follow - up of 120 cervical squamous carcinoma patients who had received operation or operation combined with radiotherapy or only radiotherapy. Methods 120 cases of therapical cervical squamous carcinoma patients were detected HPV - DNA by HC - Ⅱ and serum SCCAg using immunohistochemical method. Results The positive rate of HPV - DNA and serum SCCAg was 49.17% and 17.50% , with a significant difference between them( P 0.05 ). Conclusion HPV - DNA test with HC - Ⅱ for follow - up of cervical squamous carcinoma patients was feasible. It was more sensitive than serum SCCAg. But it suggested that high risk type HPV -DNA test combined with serum SCCAg may be the independent prognostic factors.
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Objective The prokaryotic expression vector of NADP(H)-dependent retinol dehydrogenase/reductase(NRDR)B1 was constructed for the detection of NRDRB1 protein,and to prepare its polyclonal antibodies,in order to lay the foundation to study the function of NRDRB1.Methods The coding region of NRDRB1 was constructed to the Gateway-based expression vector(pDEST 14),which was transformed into the Escherichia coli(BL21-AI)for the native protein expression.Overexpression of the recombinant was induced at mid-log growth phase of BL21-AI(A600=0.6)using 0.2% L-arabinose.After supersonication the inclusion bodies of NRDRB1 were purified.New Zealand rabbits were immunized with NRDRB1 as the immunogen,which was recovered from SDS-PAGE gel and subscapsularly injected.The titer of the antiserum was determined by dot blot assay.The antibody was purified by HiTrap Protein G column,and its activity and specificity were assessed by Western blotting and immunohistochemistry.Results The prokaryotic expression vector pDEST 14 with NRDRB1 was constructed.The constructs were sequenced by dideoxynucleotide method.NRDRB1 was overexpressed in strain BL2l-AI.The concentration of recovered NRDRB1 was 0.42mg/ml with a recovery rate of 52.3%.All the immunized rabbits produced high-titer antisera after the second booster.The titer of the antiserum was 1∶2 000 with a detection limit of 6.4ng NRDRB1.The purified antibody had a high specificity.Conclusion The present study provides an effective method of preparing polyclonal antibody against NRDRB1.The purified NRDRB1 native protein and the specific polyclonal antibody of NRDRB1 would be valuable for the study on the biological function of NRDRB1.
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Objective To analyse the microvascular density(MVD) and CD44v6 expression in uterine cervical squamous carcinoma to evaluate their clinical significance. Methods Immunohistochemical staining (LSAB method) by using antibodies against CD 34 and CD44v6 was performed on archival specimens of 50 cases of human uterine cervical squamous carcinoma and 10 cases of normal cervical tissue. Results MVD was greater in metastatic tumors than that in nonmetastatic tumors and normal control. The expression of CD44v6 was higher in metastatic tumors than that in nonmetastatic tumors and normal control. MVD and CD44v6 expressions were significantly related to lymph node metastasis(P