The Biological Effect Of Isoflavone Derivatives On Endometrium Tissue

In clinical, post-menopause women were extensively taking Hormone Therapy(HT) to improve their qualities of life. So researches reveal that HT could release the perimenopausal syndrome,protect Osteoporosis(OP) and Alzheimer's disease(AZ) which were resulted from low level of estrogen in the body. However, long term HT may increase the incidence rate of phlebothrombosis and breast cancer, and estrogen therapy without progestogen will increase the incident rate of endometrial carcinoma. The therapeutic effects of HT on releasing the perimenopausal syndrome, improving genitourinary tract atrophy and protecting OP were confirmed, but the conclusion of National Institutes of Health(NIH) from their five years study increases the caring and discussion about HT. Nowadays, researchers want to find a new Selective Estrogen Receptor Modulators (SERMs) that express estrogen biological effect with no risk of endometrial and breast cancer for the clinical drug usage.Many epidemiologic survey results showed that the rates of rectal cancer and breast cancer of post-menopause woman in Asia were lower than that in Europe, as well as the the rate of bone fracture and flush. Researches revealed the main reason is that Isoflvone(IF) in the food of Asia women is abundant. IF usually come from soy, and include Genistein (GEN), daidzein and daizeol. Although there is a little difference among these three groups, they all can be binded to Estrogen Receptor(ER), then activate the Estrogen Receptor Element(ERE) to express different estrogen biological effect. Even though the epidemiologic survey result indicate that IF can not only relieve the perimenopausal syndrome, protect OP and AZ, but also decrease the risk of breast and endometrial carcinoma, which need the large sample retrospective analysis for determination.Biological effect of IF may make it an ideal SERMs. Researches find that the ability of ligand bind to receptor determined its biological effect. The ability of IF bind to ER is weaker than that of estradiol(E2), and its estrogen biological effect is only 1/1000 of E2. It is impossible that achieving the same effect as E2 through intaking nature IF dosage. In 1988, Japan wutian company change the structure of IF to therapy OP and get good effect, which implied that the IF's biological effect can be changed after its structure has been changed.The pharmacy department of western china medical center of Sichuan university add different active group to the different parts of GEN structure and synthesis 24 IF derivatives. Early days'study showed that IF derivatives have different biological effect on different cancer. And we get four derivatives(F8,F11,ZF3 and ZF7)whose effect of inhibiting cancer cells proliferation are more powerful. All of them express estrogen effect. So now we will test their ability of inhibiting endometrial (carcinoma) cells proliferation, and hope to get the strongest IF derivative, then to apply in animal model and analysis its biological effect on endometrial tissue in vivo, which can provide theory support for explore a new SERMs.Firstly, we modified the method of primary endometrial cells culture and establish stable primary endometrial cells culture system;then analysis four IF derivatives'effect on inhibition of cells proliferation base on the culture system and Human Endometrial Cancer-1-B(HEC-1-B) though MTT test and get the strongest IF derivative; at last apply the IF derivative in animal model and test its biological effect on endometrial tissue in vivo through immunohistochemistry and western-blot methods. And the results and conclusions are as follows:1. Using enzyme digestion and two times screen filtration methods to culture primary endometrial cells;purify endometrial epithelial cells and stromal cells utilizate their volumes and the different times of adhering to hall; and apply immunohistochemistry method dentificate the culture result. The culture result showed that success in culture endometrial epithelial cells and stromal cells, which all can be passaged stably and the purity is above 90 % and 95 % .The primary endometrial cells culture system can be used by post-experiment.2. Analysis four IF derivatives'effect on inhibiting the proliferation of endometrial cells and HEC-1-B though MTT test. The results showed that ZF7 can promote HEC-1-B cells proliferation for 24 hours action; However, after 48 and 72hours, it began to inhibit HEC-1-B proliferation, as well as F11, ZF3 and F8. Their biological effects are of time and dosage dependent. On the other hand, every dosage F8, 25μM ZF3 and ZF7 act on endometrial epithelial cells for 24hours can promote cells proliferation; but on the other time blocks, all of the four IF derivatives inhibit the cells proliferation. The effect depends on time and dosage, the F11 is the strongest IF derivative in inhibiting cell proliferation.3. Intraperitoneal injection(i.i.) ovariectomy rat with F11 for ten weeks. The result show that F11 increase the ratio of uterus wet weight to body weight; stimulate rat vagina epithelium cells maturity; promote rat endometrial gland augment and make gland cave bigger. The immunohistochemistry and western-blot results show that F11 up-regulate the express of ERβin endometrium but no significant increase PCNA express, whose biological effect is dosage and time dependent. Since F11 maybe up-regulate and bind to ERβto express biological effect, and F11's effect in proliferating endometrial cells is weakly, which indicate that F11 may be become a new SERMs and need to be study further.