| ObjectiveRefering to the standard in "Traditional Chinese medicine surgery" editted in chief by Zhu Renkang ,through traditional Chinese Medicine Bianzheng, we divided the nonmelanoma skin cancer (NMSC) pacients into groups.We used general Polymerase chain reaction (PCR) and Nested Polymerase chain reaction (Nested PCR) to detect epidermodysplasia verruciformis-human papilloma virus(EV- HPV) which is some specific types of human papillomavirus (HPV) .And we sequenced amplified DNA .The aime of this study was to detect whether there is relationship between Chineae Medicine Zheng Group and HPV infection. Materials40 samples of NMSC patients came from Wuhan NO. 1 hospital clinic that were diagnosised both in clinic and in histologic.Among 40 samples ,20 fresh tissues were cut from the NMSC pacients (8 SCC pacients, 12 BCC pacients ) and stored —70℃ refigerabo immeiaterly,20 samples were come from formain-fixed,paraffin-embedded tissues(15 SCC pacients,5 BCC pacients). Method1. Dividing into groups:Refering to the standard in "Traditional Chinese medicine surgery" editted in chief by Zhu Renkang ,through traditional Chinese Medicine Bianzheng , we divided the nonmelanoma skin cancer (NMSC) pacients into four groups,which are Chuangganfengdu 11 ,Ganhuoxuezao 6,Yuanqixuruan 15 and Ganshenkuisun 8. 2.DNA extraction in the fresh tissues: ①3-5g tissue specimens were minced and treated 24 hours with 30ul proteinase K (10 mg/ml) and 200ul No.1 buffer at 37℃.After 24 hours,added in 30ul proteinase K (10 mg/ml) again and went on treating 24 hours.That is rough DNA. ②The rough DNA is purified by Phenol, Chloroform, isoamyl alcohol, Rubbing alcohol and ethanol. 3.The fresh tissues DNA PCR amplification: CP65-CP70 and CP66-CP69 are the primers that are specific for the detection of the whole range of EV-associated papillomaviruses. The CP65-CP70 nucleotide sequence of the selected 59 primer was 5' CA(A/G) GGT CA(C/T) AA(C/T) AAT GG(C/T) AT3' (CP65) and that of the 3' primer was 5' AA(C/T) TTT CGT CC(C/T)A(A/G)A G(A/G)A (A/T)AT TG(A/G) TC 3' (CP70), and the annealing sites of these sequences corresponded to positions 6832 to 6851 and 7273 to 7298 in the HPV-8 genomic sequence, respectively. The CP65-CP70 amplifies a 452- to 467-bp product, depending on the target HPV type. The CP66-CP69 nucleotide sequence of the 59 nested primer was 5' AAT CA(A/G)(A/C)TG TTT (A/G)TT AC(A/T) GT 3' (CP66) and that of the 3' nested primer was 5' G(A/T)T AGA TC(A/T) ACA T(C/T)C CA(A/G) AA 3' (CP69). Their respective annealing sites were at positions 6862 to 6881 and 7231 to 7250 in the HPV-8 genomic sequence. The CP66-CP69 amplifies a about 370bp product.4. DNA extraction in the formain-fixed,paraffin-embedded tissues:①First cut about 10um formain-fixed,paraffin-embedded tissues,then treated 72 hours with 30ul proteinase K (10 mg/ml), 500ul TES buffer and 20%SDS at 56℃.Add 30ul roteinase K (10 mg/ml) other 24 hours. Heat inactivation of the proteinase K (10 min at 95℃). That is rough DNA.②The rough DNA is purified by Phenol, Chloroform, isoamyl alcohol, Rubbing alcohol and ethanol.5. The formain-fixed,paraffin-embedded tissues DNA PCR amplification:The degenerate primer FAP59/FAP64 designed from two relatively conserved regions of... |
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