The Experimental And Clinical Research On Cervical Local Immune Microenvironment With Zedoary Oil Intervention

In the female cancers,the cervical cancer is the second in number and first in developing country.Nowadays,according to the researches in China and abroad,there is a very close relationship between the occurrence and development of cervical cancer and the cervical local immune microenvironment.Some documents show that there are some CTL and Treg cells in focuses of some CIN or cervical cancer patients.The documents also show that Treg cells involve in anti-tumor immune response in cervical lesions.Meanwhile,the documents find that when PD-1and CTLA-4 on CTL cell surface are overexpressed,CTL cell will stay in immunologic arrest.Just the immunologic arrest disrupts the balance of immunity and accelerates the development of tumors.Because of the findings above,the immunotherapy of tumors gets more and more attention.Zedoary oil is one of traditional Chinese herbal medicine which main component is curcuma aromatic oil.Some experts found that zedoary oil has great controlling effect on ovarian carcinoma cells and endometrial cancer cells but there are few studies on its effect on cervical cancer.Just for the research conditions,this article will be divided into two parts.The first part will discuss the regulation effect of zedoary oil on the cervical local immune microenvironment on the level of cell and molecular.And the second part will estimate the actual curative effect of CIN through comparing the expression of protein of Ki67,P16,P53 in cervical diseased tissue before and after the treatment.The study of zedoary oil's mechanism on anti-virus and anti-tumor will make theoretical basis for the clinical application.I the zedoary oil's regulation on SiHa-Treg-CTL systematic immune microenvironmentTarget:taking co-cultured system of SiHa supernatant fluid of cervical cancer,CTL and Treg lymphocyte as target,this part will discuss the zedoary oil's effect on the cervical local immune microenvironment through observing zedoary oil's influence to CTLA-4 and PD-1 protein expression under the conditions of Treg.Method:1.Separate CD8+T cell of human peripheral blood with magnetic activated cell sorting(MACS)and estimate the purify of the separated cells with flow cytometry(FCM);separate Treg cell of human peripheral blood with magnetic activated cell sorting(MACS)and estimate the purify of the separated cells with flow cytometry(F CM)2.Estimate SiHa nutrient solution's effect on ratio of CD8+T cell and Treg;3.Estimate the effects on multiplication of CTL caused by different-concentrate zedoary oil solution with CCK8;4.Estimate the CTLA-4 and PD-1's protein expression in the follow five system:CTL culture group(control group),CTL+Treg co-cultured group(Treg group),CTL+75%SiHa culture supernatant(tumor supernatant),CTL+Treg+75%SiHa culture supernatant(co-cultured group),CTL+Treg+75%SiHa lu/ml zedoary oil water solution extracted by alcohol(zedoary oil)and compared the results.results:1.The purity of CD8+T is 89.37±0.83%and of CD4+CD25+T is 82.12±2.17%.the purity comes from their separation with MACS and test by FCM2.Through Comparing the different-concentrate SiHa cultured supernatant's effect on CD8+CTL cell with blank serum's,the 75%concentration cell cultured supernatant has statistical differences(P<0.05);comparing 50%concentration cell cultured supernatant effect on Treg with 75%concentration,there is statistical difference(P<0.05).According to the comparisons above,75%concentration SiHa cultured supernatant will be the normal one in following tests.3.1ug/m1?1.5ug/ml?2ug/ml zedoary oil water solution extracted by alcohol can accelerate the multiplication of CTL cells but 1ul/m1one has the best result.So lul/ml zedoary oil water solution extracted by alcohol will be normal drug level in following tests.4.From the results of Western-Blot electrophoresis stripe gray-scale value,the protein expression of CTLA-4 and PDI is increased in CTL+Treg co-cultured group.After the interruption of zedoary oil,the protein expression of them decreased obviously.The result shows that zedoary oil can inhibit the Treg's promotion effect on CTLA-4 and PD-1.Conclusion:Zedoary oil can change local immune microenvironment by decreasing the protein expression of CTLA-4 and PD-1 and on that base,can inhibit the development of tumor.? zedoary oil's effect on immunity-related transcription factor in CIN tissueTarget:analyze zedoary oil's pharmacologic action mechanism through comparing the protein expression of Ki6,P16,P53 in CIN tissue before and after the treatment.Method:choose all the 30?40 aged patients diagnose as CIN in diagnostic sense in Hebei United University Affiliated Hospital.All of them was divided three groups although they reserved uterine and fertile function with unclear cutting edge and refused to take the supplemental operation.The first group,31 patients,underwent LEEP(Loop Electrosurgical Excision Procedure),the second,33patients,and third group,34 patients,underwent cervical cold knife conization.After their operation,the protein expression of Ki67,P16,P53 in cervical tissue is tested.All the patients were requested to use one zedoary oil suppository,1.74g/softgel,in cervix uteri every night since the seventh day after operation and stop use it during menstrual period.The treatment lasted three months.After the three-month treatment,the protein expression of Ki67,P16,P53 was tested again and compared the result of two experiments.Results:The P16 protein expression is decreased in all patients after using zedoary oil as a treatment.And the difference has statistical sense.The protein expression of P53,Ki67 of the second and third group patients decreased which difference has statistical sense while comparison result of the first group has not statistical sense.Conclusion:The protein expression ration of P16,P53,Ki67 will be decreased effectively after the zedoary oil treatment.Zedoary oil has some certain effect on CIN therapy.