Small Hairpin RNA Targeting Inhibition Of NF-κB Gene In Endometriosis Therapy Of Macaca Fascicularis

BackgroudEndometriosis is a common disease which frequently occurred in women of reproductive age,10 to 15% of women may have a chance to suffer this disease. The stigmata of endometriosis include dysmenorrhea, dyspareunia, chronic pelvic pain, irregular uterine bleeding, and/or infertility, which seriously affected women’s health and quality of life. As with the improvement of the healthy consciousness, the morbidity trend towards higher year by year. Since the endometriosis first reported in 19th, the pathogenesis still was a mystery, although experienced more than 100 years’ reseach. Among those different theory, such as endometriosis cultivation theory, coelomic metaplasia theory, lympatic and venous spread theory, as well as the theory of stem cell, vascularization, eutopic endometrium determinism, endocrine immune theory, etc, all these theory can not figure out the pathogenesis thoroughly. However, all the ectopic endometrium tissues which keep survival should go through three steps: adhesion, invasion and angiogenesis. Depending on this theory, if we can block up one more of the three steps, the occurrence and development of endometriosis can be inhibited.Although the endometriosis is a benign disease, it has the malignant tumor behaviors with implantation, invasion, proliferation, metastasis and recurrence. For example, the ectopic endometriosis tissues can be transferred in the abdominopelvic cavity, and then forming new lesions. At present, the main treatment forcus on endometriosis are surgery and drug therapy, but the outcome of these methods are unsatisfactory because of the recurrence. Therefore, a new therapeutic method is imperative. Nowadays, the Gene therapy has become a new therapeutic strategy. NF-κB, as a nuclear transcription factor, was first founded in the B-lymphocytes. Because it can be combined with the IκB, so it was named NF-κB. The transcription factor NF-κB is a family of peptides formed by five subunits:RelA or p65, RelB, c-Rel, p50/p105, and p52/p100. These subunits bind forming different NF-κB dimers that remain inactive by their union to the NF-κB inhibitory protein IκB in the cytoplasm. Once actived by activator, the I κ B will deperated from the NF-κB dimers. As a result, NF-κB free dimers are able to bind to DNA, initiating NF-κB target genes transcription. Until now, NF-κB has been associated with the physiological and pathological processes of cell proliferation, angiogenesis, inflammation, cell apoptosis and plays a very important role. It has been reported that the factor has the certain relevance with the development and prognosis of breast cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, etc. It has also reported that NF-κB was involved in the pathophysiology process of endometriosis. Our research team also confirmed that NF-κB plays an important role in the development of endometriosis in pre-experiments in nude mice. After inhibiting NF-κB gene expression, ectopic lesions significantly atrophy, as well as reduced microvascular density. Therefore, the primates were used as the experimental subjects. Through the RNA interference technique, targeted to inhibit the expression of NF-κB gene by short hairpin RNA. Then, the effect on the development of endometriosis lesions was observed, a preliminary assessment of the therapeutic effect and safety on endometriosis was done after the experiment finished.Objective1. Establishing the animal models of endometriosis in macaca fascicularis through surgery in vitro, meanwhile, the effect of the endometriosis model was observed, in addition, more options for choosing the endometriosis model in large primates was provided.2. The endometrium cells of macaca fascicularis was cultrued in vitro, while the high specificity adenovirus vector-mediated shRNA targeting NF-κB was synthetised. Then the endometrium cells of macaca fascicularis was interfered with the adenovirus Aafter that, the number of apoptotic cells and the expression of apoptosis proteins were compared between the experimental and control groups, as a result, a assess was made in weather there has a direct inhibitory or not in the proliferation activity of endometrium in macaca fascicularis.3. After successfully established the endometriosis model of macaca fascicularis, we directly inject the NF-κB shRNA adenovirus which pre-synthesized into ectopic lesions. Through targeting inhibition the expression of NF-κB gene in ectopic lesions, we observed the different expression of microvascular density, proliferating cell nuclear antigen and the NF-κB gene among experiment group, negative control group and control group. At last, we assess the effect on the treatment of endometriosis in macaca fascicularis through targeting inhibition the expression of NF-κB gene in ectopic lesions, and then its long-term side effects were observed. Sacrificed all the macaca fascicularis after six months, long-term side-effects was observed in variously important organs.Methods1. Fifteen female macaca fascicularises which were healthy and weight similar with regular menstrual cycles were selected, they were divided into experimental group and the control group,12 for experiment group,3 for control group. Six menstrual cycles was observed aiming to remove the unhealthy macaca fascicularises.2. By the time of the first 8-15 days of the menstrual cycle or between the third and fifth day of estrogen peak, a surgery was done to get endometrial tissues in experiment group, which were implanted into the pelvic omentum majus, Douglas pouch, as well as uterus surface during the surgery. The same surgery was undergoed on the control group, but the omentum majus was implanted instead.3. An exploratory surgery was done by laparoscopic at the time eighth and sixteenth week of post-operation respectively, then a histopathological examination of suspicious lesions was took. As a result, weather there has endometrial glandular structures and mesenchymal cells in suspicious lesions were observed under the microscopic.4. Designed the NF-κ B-p65-shRNA adenovirus and the corresponding negative control adenovirus, then synthetized the high specificity adenovirus vector-mediated shRNA targeting NF-κB.5. First, we received the fresh endometrial cells of macaca fascicularis by surgery, then, the endometrial cells were cultured in vitro, and these cells were randomly divided into experimental group and control group. The experimental group was transfected the NF-κB-p65-shRNA adenovirus and the negative control adenovirus to the control group simultaneously. The changing indications of cell proliferation activity were observed after co-culture, such as the expression of the target gene protein and apoptotic protein, the situation changes of the cell proliferation and cycle.6. The successful established endometriosis model of macaca fascicularis, the the high specificity adenovirus vector-mediated shRNA targeting NF-κB and the corresponding negative control adenovirus were readied before the next experiment.7. Selected the macaca fascicularis model of endometriosis, which divided into experimental group, negative control group and simple model group. The high specificity adenovirus vector mediated shRNA targeting NF-κB and negative control shRNA adenovirus with no-load NF-κB gene were synthesised. The experimental group injected the adenovirus which carried the NF-κB shRNA into the endometriosis lesions under Laparoscopy surgery, the negative control group with no-load shRNA adenovirus and the simple models group injected with normal saline.8. Four weeks later after the injection, an observed operation was performed through laparoscopic and some lesions were collected. The CD34 immunohistochemistry of these lesions were done to detect the microvessel density, then the variation of the microvessel density among each group were observed. The expression of the NF-κB gene and PCNA (Proliferating Cell Nuclear Antigen) were detected through western blot.9. Statistical analysis:all of the experimental quantitative data was described with the arithmetic mean ± standard deviation (X± SD).The differences of means in groups was compared by One-way ANOVA. Independent samples t-test was used to compare the differences between the treatment group and control group, α= 0.05 was the inspection standard, namely, P<0.05 was considered statistically significant.Results1. Preoperative observation of the macaca fascicularis on mental status, appetite, sleep, and the menstrual cycle:through observed nearly six menstrual cycles, we found that all macaca fascicularis have a good spirit with no lethargy and lazy sleepy phenomenon. Meanwhile, the macaca fascicularis’s sleep and appetite were good. All macaca fascicularis have a regular menstrual cycle of 28-30 days, with a menstrual phase of 4-5 days, which was similar to women of childbearing age.2. On the eighth week after the surgery, Exploratory in pelvic revealed that five of them were found suspected ectopic endometrial lesions in the receptor site among fifteen experimental macaca fascicularis, five with the chocolate cysts, the other two with no ectopic lesions exist. Suspicious lesions histopathological examination revealed the complete structure of the glands and stromal cells. On the other hand, we did not see any suspicious lesions in control group, as well as no complete structure of the glands and stromal cells were found in histopathological examination for the tissues. Laparoscopic exploration was performed again after sixteen weeks, endometriosis lesions still can be found, and there are different degrees of pelvic adhesions. The ectopic endometrial growth was similar to the first time.3. We successful synthetize the high specificity adenovirus vector-mediated shRNA targeting NF-κB, Adenoviral vector sequencing results were consistent with the expected results, the virus titer after packaging was 1.58 × 1011 U/ml.4. After infecting endometrial cells with the NF-κ B-p65-shRNA adenovirus, the experimental group has a higher apoptotic proteins expression levels compared with the negative control group, the difference was statistically significant. What’s more, the experimental group has a low number of cells stayed in division stage compare with the control group, which means the cell proliferation was suppressed.5. The experimental group has an evident atrophy in ectopic lesions compared with the previous, the lesions’ microvessel density in experimental group decreased evidently compared with the negative control group and simple model group (0.00198±0.0003 versus 0.02189±0.0026 versus 0.02451±0.0033), and the differences was statistically significant (P<0.01). The expression of PCNA (0.37±0.17 versus.57±0.26 versus 0.57±0.28) and NF-κB (0.338±0.174 versus 0.678±0.021 versus 0.645±0.098) in experiment group was lower than the negative control group and simple model group, the differences were statistically significant (all P<0.01). Conclusions Through targeting suppressed the NF-κB gene expression by NF-κB shRNA, we can inhibit the development of endometriosis through reducing the ability of angiogenesis and cell proliferation of ectopic endometrial cells.Conclusion1. According to the implantation theory, we successfully established the endometriosis model of macaca fascicularis for the first time in China, which provides a theoretical basis for endometriosis research in the field of non-human primate model especial on the macaca fascicularis. In addition, it added a new animal choice on the research of endometriosis in non-human primate field. Not all the macaca fascicularis endometriosis model can be successful established suggested that the differences of the individual genetic, immunity and other factors may influence the occurrence of endometriosis.2. The NF-KB-p65 shRNA adenovirus can effectively accelerate the apoptosis of endometrial cells and inhibit the proliferation of endometrial cells, which provide the theoretical basis for the future study in the macaca fascicularis model of endometriosis about NF-κB.3. Through targeting suppressed the NF-κB expression by NF-κB shRNA, we can inhibit the development of endometriosis through reducing the ability of angiogenesis and cell proliferation of ectopic endometrial cells.