| IntroductionOne of the two X - chromosomes in cells of female is randomly inactivated in the early stage of embryological development, results in random X - chromosome inactivation pattern of tissues of normal female. Theoratically neoplastic tissues are composed of clonal proliferation of a malignant cell and their X - chromosome inactivation pattern should be nonrandomly. X - inactivation analysis is the best method for clonal analysis of human neoplasia. HUMARA. ( human androgen ?receptor gene) analysis is most used for X inactivation analysis. Our study assessed the clonality of cervical cancer and cervical intraepithe-lial neoplasia ( GIN) with the popurse to afford a new molecular biological method for the research of cervical cancer and GIN.Material and MethodCase collection. Fresh cervix tissue samples were collected from cases undergoing cervical biopsy procedure or surgical in the Second Hospital of China Medical University and Liaoning Province Tumor Hospital from July 2001 to January 2003. Among them are trial group with 26 invasive cervical cancers (9 in stage 1,10 in stage II and 7 in stage III) and 31 CINs(12 CIN Is, 10 GIN Us and 9 GIN Is)and control group wih 33 normal cervixes. After collection, samples wereimmediately stored at - VOt for subsequent experiment. Each case is verified by pathological diagnosis.Extract tissue DNA with routine phenol - chloroform - isoamyl alcohol method.Restriction enzyme treatment. 10μl DNA solution was added to two Eppendorf tubes respectively with trial tube containing Hhal 2μl (10U/μl) , 10 × M Buffer 4μl, distilled water 4μl and control tube containing 10 x M Buffer 4μl, distilled water 6μl and incubated at 371 overnight. Reaction was stopped by heating the mixture at 70# for 20 min.PCR amplification. Primer sequences for PCR were 5 ' - TCC AGA ATC TGT TCC AGA GCG TGC -3and 5'- GCT GTG AAG GTT GCT GTT CCT CAT - 3 ' Amplificated fragment length was 255 -315bp. 25μl of PCR reaction contained 5μl of template DNA, 0.2μl of each primer (0. 1ng/μl) , 2μl of 2. 5mM dNTPs, 0. 2μl of Taq polymerase, 2. 5μl of 10 × Buffer and 14.9μl of dd.H2O. 40 cycles of amplification were carried out using cycling parameters of 94℃ for 45s, 60℃ for 1 min, 72℃ for 1 min with initial 94℃ for 3 min. PCR products were electrophoreased at 200 V for 4 - 5 h.Interpretation of clonality data. Cases were considered as heterozygous (informative) if PCR amplification of undigested DNA showed two major bands with equal intensity. Cases with only one major band were homozygous for HUMARA and thus uninformative. For informative cases, after restriction enzyme digestion, monoclonality was indicated when PCR amplification showed two bands with signal intensity ratio > 0. 3 and poly clonality was indicated when PCR amplification showed one band or two bands with signal intensity ratio ≤0.3.Statistical Analysis. Statistics was made with software SPSS11.0. Differences among the groups were analyzed by ^ test or Fisher s test.Results1. Heterozygotic rate of HUMARA was 88.9%.2. Monoclonality of cervical cancer, GIN and normal cervix were 91.7% , 38.5% and 10% respectively. Differences among them were statistically significant ( P < 0.05 ).3. Monoclonal rate of cervical cancer in stage I, II, III was 85.7%N90% and 100% respectively. Differences among them were no statistically significant ( P > 0.05 ).4. Monoclonal rate of GIN l^ll^ III was 20% ^ 37. 5% and 62.5% respectively. Differences among them were no statistically significant (P>0.05).DiscussionClonality assessment and HUMARA analysis. One of the two X -chromosomes in cells of female is randomly inactivated in the early stage of embryological development, results in random X - chromosome inactivation pattern of tissues of normal female. Theoratically ne-oplastic tissues are composed of clonal proliferation of a malignant cell and their X - chromosome inactivation pattern should be nonrandom. X - inactivation analysis is the best method for clonal anal...
|
PDF Full Text Request