Detection Of Human Papillomavirus DNA And Its E Gene Mutation In Cervical Cancer Tissue

OBJECT: (1) To explore an effective method for the detection of human papilloma virus DNA in cervical cancer tissue. (2) To study the relationship between the detection rates of high-risk HPV types and the clinical diagnosis of cervical cancer. (3) To test the significent of HPV-DNA amplification in finding pelvis lymph node in cervical cancer. (4) To explore the intratypic nucleotide and amino acid sequence variations in the E6 and E7 open reading frame of human papillomavirus type16 in cervical cancer tissue in GuangDong.METHODS'. (1) HPV L1 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR with general consensus primers. The types of HPV was identified by PCR product sequencing. (2) HPV L1 gene fragment in cervical cancer tissues was amplified by HPV-specific PCR with general consensus primers. The types of HPV was identified by PCR product sequencing. (3) HPV L1 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR with general consensus primers. The types of HPV were identified by PCR product sequencing. The relationship between the detecting of HPV and the pathological alternation in the pelvis lymphatic node of cervical cancer was analyzed. (4) The types of HPV was identified by PCR product sequencing. HPV16 E6 and E7 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR. The intratypic nucleotide and amino acid sequence variations was found by DNAsis identify. The changes in antigenicity was identified by Antigenicity ValueX10.RESULTS'. (1) The positive rates of high-risk HPV types in 50 cases of cervical cancer is 78%. And the mixed infection with HPV16 and HPV18 was the most common, accounts for 48%. Addtionally, type 58 HPV was detected in one case. Sequencing results showed no difference of LI sequence between the sample and German standard strain. (2 ) The detection rates of high-risk HPV types in 50 csaes of cervical cancers is 78%. And the mixed infection of HPV16 and HPV18 is most common, accounts for 48%. Addtionly, type 18 HPV was detected in 2 patients, and type 16 HPV in 1 case, while mixed infection with HPV 16/18 seen in 2 cases. (3 ) The positive rate of HPV-DNA detection in the pelvis lymph node from 40 cases of cervical cancer was 40%. HPV-DNA was detected in each of the 10 cases of cervical cancer with pathological changes in pelvis lymphatic node. Addtionally, HPV 18 was detected in both of cervical and pelvis lymphatic node in some cases. (4 ) The positive rate of high-risk HPV types in 50 csaes of cervical cancers was 78%. And the mixed infection with HPV 16 and HPV 18 was found in 18 cases. The infection with HPV 16 only was seen in 15 cases. HPV16E6 and E7 sequences were amplified from 25 cases of total 33 HPV-positive cases. The 178th neocleotide in the viral DNA sequence "A" was changed into "C" , which caused a Asp cordon of E6 gene to be replaced by a Glu cordon. The 647th neocleotide in the viral neocleotide sequence "T" was changed into "C", and that caused a Asn cordon of E7 gene to be replaced by a Ser cordon.CONCLUSIONS: (1) PCR-Direct sequencing approach was an effective way for the detection and typing of human papillomavirus DNA in cervical cancer tissue, through which we could find the rare special types. At the same time, it can also showed the mutation of HPV DNA sequence. (2) The mixed infection of HPV 16 and HPV 18 might more easily induce the pathogenesis of cervical cancer. The type of HPV18 might be more dangerous than the type of HPV 16. ( 3) HPV-DNA could be detected earlier than the pathological changes. The detection of HPV-DNA could be helpful in finding the pelvis lymph node metastases in cervical cancer.lt is a sign for bad precaucion. HPV 18 is the poor type for it was found metastases early. (4) By sequencing and phylogenetic analysis, the most frequently observed substitution in HPV16E6 ORF wasdistributed in the discrete regions:94-241. Some substitutions resulted in non-silent mutation. The mutation hot-spot of HPV16E6 in cervical cancers from Guangdong was found at neolertide 178 in the DNA sequence. The most frequently observed substitution in HPV16E7 ORF was distributed in the discrete regions:647-846. Some substitutions resulted in non-silent mutation. The mutation hot-spot of HPV16E7 in cervical cancers from Guangdong was found at neolertide 647 and 846 in the DNA sequence.