| Objective1.To investigate the clinical significance of the positive expression of human telomerase reverse transcriptase(hTERT)mRNA in diagnosing squamous carcinoma of the cervix (SCC) and cervical intraepithelial neoplasia (CIN) by detecting the positive expression of hTERTmRNA in exfoliatied cells from samples extracted from SCC and CIN and comparing with that of normal cervical exfoliated cells.2. To determine the relationships between the severity of cervical lesions and quantitative expression levels of hTERTmRNA by detecting the quantitative expression levels of hTERTmRNA in exfoliated cells of SCC and CIN and comparing with that of normal cervical exfoliated cells.3. To investigate the interaction of the positive rate of infection with high-risk human papillomaviruses (HR-HPV) in exfoliated cells of SCC, CIN and normal cervical positive expression levels of hTERTmRNA of the samples with regards to occurrence and development of cervical canceration by detecting, comparing and conducting a correlation analysis between the two relevant factors.Materials and Methods:1.Research materials:The Hela cell strains of cervical carcinoma and cervical exfoliated cells from a collection of both clinically and pathologically confirmed cases of SCC(n=31), CIN II～III(n=26), CIN I(n=22)and normal cases or cases of chronic cervicitis (n=21) in Guangdong Women and Children's Hospital (GDWCH) affiliated to Guangzhou Medical College from April 2008 to December 2008. Samples of normal or chronic cervicitis were extracted from the clients in GDWCH who underwent panhysterectomy due to uterus myoma or uterine prolapse. 2.Methods:①Cell culture ,collection and preservation: Hela cell strains were maintained on purified DMEM medium containing 10% bovine serum, collecting Hela cells at 1×106 copies and preserving at -80℃. Cervical exfoliated cells: double specimens of cervical exfoliated cells were collected, one was washed with RNase-free PBS, frozen in RNA conservation buffer at -20℃or extracted immediately.The other was processed with hybribio collection and conservation system to treat the DNA specimens of cervical cells.②The real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)was employed to detect the hTERTmRNA positive expression and quantitative levels of cervical exfoliated cells in Hela cell strains, SCC, CIN II～III, CIN I as well as normal or chronic cervicitis,the latter was expressed in manner of relative quantitation . Taking the number of hTERTmRNA expression levels in Hela cells as the basis, the quantitative levels of hTERTmRNA of each positive sample was counted as the relative numbers as NhTERT. Employ HybriMax techniques to detect positive rate of HR-HPV infection in exfoliated cells from SCC,CIN II～III,CIN I as well as normal or chronic cervicitis.③Statistical analysis: Using SPSS16.0 to analyze the data, Fisher's exact test to assess sample rate comparison,non-parametric Rank Kruskal-Wallis H to compare the sample mean,linear and Spearman Rank Correlation to conduct correlation analysis, considerα< 0.05 bilaterally as statistical significance.Results1.The positive rate of the hTERTmRNA in cervical exfoliated cells from SCC , CINII～III , CIN I and normal cervix were expressed as 80.6% ,53.8%, 27.3% ,and 9.5%, respectively. The positive expression rate of hTERTmRNA in SCC, CINII～III exfoliated cells were significantly higher than that of normal cervix, indicating statistical significance (P < 0.001, P = 0.002).2.Quantitative levels of hTERTmRNA(NhTERT) in SCC ,CINII～III, CINI and normal cervical exfoliated cells respectively were expressed as 0.4646～13.7823, 0.2278～3.5000, 0.0459～0.5480, 0.0089～0.0106 , indicating statistical significance (P < 0.001) after rank-sum test comparison between the four groups. NhTERT increases in accordance with the severity of the cervical disease, after using Spearman rank correlation analysisγ= 0.676,(P<0.001), demonstrating the hTERTmRNA expression levels present significant positive correlation with the severity of the cervical disease.3. The positive rate of HR - HPV in SCC, CINII～III, CIN I and normal cervical exfoliated cells were expressed as 93.5%, 76.9%, 54.5% and 4.8%, respectively.The percentage of hTERTmRNA expression and HR-HPV infection showing simultaneously positive results as 77.45% and 42.6% separately, which is 4.8% higher than that of normal cervical cells, indicating statistical significance (P< 0.001,P=0.002).Positive correlation is also found betweeNhTERTmRNA positive expression and HR-HPV infection(γ= 0.392,P< 0.001), indicating statistical significance (P < 0.001, P = 0.002).Conclusions1.The hTERTmRNA positive expression rate is higher in exfoliated cells from SCC and CINII～III than that of normal cervical cells,indicating that the detection of the hTERTmRNA positive expression of cervical exfoliated cells could clinically assist the early diagnosis of pathological changes as CINII and above.2.An obvious positive correlation between the quantitative expression levels of hTERTmRNA in exfoliated cells of SCC,CINII～III and CIN I and normal cervix with the severity of the cervical lesions, indicating the positive expression levels of hTERTmRNA presents close relationships with the development of cervical canceration.3. Expression levels of hTERTmRNA in exfoliated cells of cervical lesions demonstrate a positive correlation with HR-HPV infection, implicating that HR-HPV plays a role in regulating and controlling the expression of hTERTmRNA and the activation of telomerase, resulting in one possible carcinogenic mechanism.4.An infection with high-risk HPV in cervical exfoliated cells from cervical severe diseases is much higher than that of normal cervix. The expression of hTERTmRNA is positively correlated with the infection with high-risk HPV and severity of cervical disease . |
