Incidence Of JAK2V617F Mutation In Patients With Myeloproliferative Disorders And The Effects Of Imatinib,Interferon,Garlicin On Proliferation, Apoptosis And Gene Expression Of HEL Cells

Objective: 1.Detecting the occurrence of JAK2V617F mutation in the patients of MPD and MDS 2.Studying the effect of growth,apoptosis,gene expression when the HEL cells were cultivated in different concentrations of Imatinib, Interferon and Garlicin.Methords :The study included two parts.In the first part,there were 46 patients with MPD, including 26 PV and 20 ET. Another group is 9 patients with MDS. Control group were 10 healthy volunteers. The experimental procedure:1 Extract genomic DNA; 2. Perform Allele-specific PCR and amplification of JAK2 exon14. 3. Collect PCR products for agarose gel electrophoresis and select the positive products of JAK2V617F mutation in patients for sequencing. 4.Detect peripheral blood cell count between JAK2V617F-positive and negative patients.In the second part, HEL cells were cultivated in the RPM1640 medium with diferent drug concentration. MTT was used to detect their effect on cell proliferation. Flow cytometry was operated to detect apoptosis by using PI single staining. Fluorescence quantitative PCR was detected to observe the levels of JAK2V617F expression.Results:Among the 46 patients with Myeloproliferative disorders , the total positive rate was 67.39%, 20/26(76.92%) with PV,11/20(55.00%) with ET,2/9(22.22%) with MDS. The positive rate of JAK2V617F mutations in peripheral blood and bone marrow were equal in each group. JAK2V617F positive was not found in 10 healthy volunteers. The sequencing showed that the mutation sites were the same. The peripheral blood white blood cell and Hemoglobin count of JAK2V617F positive patients of PV were significantly higher than that of negative patients. The platelet count of JAK2V617F positive patients were higher than that of negative patients,but there was no significant difference. The peripheral blood white blood cell count of JAK2V617F positive patients of ET was significantly higher than that of negative patients.Hemoglobin count of JAK2V617F positive patients of ET was higher than that of negative patients, but there was no significant difference. The platelet count of JAK2V617F positive patients were lower than that of negative patients, there was no significant difference.Imatinib,Interferon and Garlicin could all inhibit the proliferation of HEL cells, stronger inhibition with the increase of drug doses. At cultivating 72 hours, Inhibition rates were significant differences in groups of different concentration Imatinib (P<0.05). In Interferon group, Inhibition rates were significant difference (P<0.05) except between 5×105u/L group and 10×105u/L (P=0.251) groups. In Garlicin group, Inhibition rates were significantly difference (P<0.05) except between 5mg/l group and 10mg/L group(P=0.145). Interferon and Garlicin but STI571 could induce to apoptosis to HEL cells obviously with the increase of concentration. Garlicin induced a higher rate of apoptosis at a lower concentration. The apoptosis rate reached 88.54±2.92 in 50mg/L concentration.The JAK2V617F expression of HEL cell declined with cultivating prolonged in the groups of Interferon and Garlicin.To cultivate HEL cells for 24h,48h and 72h, the fluorescence quantitative data of JAK2/GAPDH was 1.556, 1.213 and 0.870 in 100×105u/L concentration of interferon group, being 1.556, 1.213 and 0.870 in 10mg/L of Garlicin gouup. However, the fluorescence quantitative data of JAK2/GAPDH was 1.016,2.019,3.022 in 20.0umol/L of Imatinib group. Conclusion: 1.The incidence of JAK2V617F mutation in MPD patients was very high in MPD patients.The positive rate in PV patients was the highest,ET followed.Also,there was JAK2V617F point mutation in MDS.2. The positive rate of JAK2V617F point mutation in peripheral blood and bone marrow was equal in MPD.3. JAK2V617F detection contributed to the diagnosis of MPD. The peripheral blood white blood cell and Hemoglobin count of JAK2V617F positive patients of PV were significantly higher than that of negative patients. The peripheral blood white blood cell count of JAK2V617F positive patients of ET was significantly higher than that of negative patients.4.Imatinib, Interferon, Garlicin could all inhibit proliferation HEL cells with JAK2V617F mutation. 5 Inducing apoptosis and inhibiting the expression of JAK2V617F mutation may be the mechanism of Interferon and Garlicin.