| Part one The Improvement of Human Pituitary Adenoma CultivationBackgoundPituitary adenoma is a kind of endocrine tumor, its morbidity rank the third in intracranial tumors, after glioma and meningioma. For pituitary adenoma is a benign tumor, primitive culture is difficult and slow. The main problem is that because the culture system of pituitary adenoma often mixed with a large number of fibroblasts. After a period of time, the cultivation system appeared in a large number of fibroblasts and even replace pituitary tumor cells. And no pituitary tumor cell lines, in the past few decades, scholars all over the world on the basis of the research model used rat pituitary tumor cell line instead, such as GH3, ATT20, etc. Long-term, stable, high purity way of human pituitary adenoma culture have always been many scholars goal of research.In pituitary adenoma culture research, Ilona Fazekas’years cultivation of pituitary adenoma cells and summarizes the characteristics of different types of pituitary adenomas develop in which the growth hormone adenoma type and ACTH adenoma relative to cultivate more easily, and prolactin type adenoma and non-functional adenoma relative to cultivate more difficult. GH adenomas, ACTH adenoma and eosinophilic stem cell adenoma are stick wall to grow and migration and growth better. PRL adenomas and non-functional adenoma have poor activity of proliferation. They grew slower and rare and difficult to stick to wall and have irregular nucleus, cytoplasm in organelles, their hormone secretion granules is smaller.Ishiwata fiber cells is difficult to separate with pituitary adenoma cells in the cultivate process. Without separation of fibroblasts, the fibroblasts growth, gradually replace pituitary adenoma cells in the culture system, lead to failure. But at the same time, some scholar thinks, fibroblasts on pituitary tumor cells initially a trophoblastic role but in the end will limit the growth of pituitary tumor cells. How to suppress fibroblasts has been the hotspot in the research field of the pituitary adenoma culture. For this problem Auda-Boucher G and others tried to use D valine in culture medium, thereby inhibit the growth of fibroblasts.Domestic research mainly include Xu Weiguang and ShouXueFei scholars study of pituitary adenoma cultivating mode, most scholars have advocated the pituitary adenoma with multiple poly lysine, adhesion in the bottom of the dish, monolayer culture, because monolayer culture in the process of changing culture media was not easy to loss cells, and cell tile in the bottom of the dish, is advantageous to the observation of cell morphology and structure. But pituitary adenoma with poly lysine adhesive in the bottom of the dish, fibroblasts were difficult to separate from pituitary adenoma, cannot achieve the purpose of maximum inhibit fibroblasts.According to the tumor diameter size classification,1 cm or less called micro adenoma,1～4 cm for large adenomas,> 4 cm for large adenoma. And giant pituitary adenomas often involving unilateral or bilateral cavernous sinus area, by surgical resection and protect its corresponding nerve function integrity, relatively difficult, at this moment need drugs or stereotactic reflection treatment, to achieve therapeutic purposes. Pituitary adenoma and depending on the type of pathology classification can be divided into 7 kinds, growth hormone (GH) cell adenomas, prolactin (PRL) cell tumor, adrenocorticotropic hormone (ACTH) cell adenomas, thyroid stimulating hormone (TSH) cell adenomas, gonadotrophin (LH/FSH) cell adenomas, mixed neoplasia, non-functional adenoma. Prolactin adenoma and growth hormone adenoma can effective drugs for medical treatment is still exist, but other types of pituitary adenomas, especially non-functional pituitary adenomas, there is no effective drugs.Pituitary adenoma originated in ectoderm, belongs to the epithelium, and fibroblasts are often originated from mesoderm origin of interstitial tissue. Cytokeratin (cytokeratin, CK) mainly distributed in epithelial cells, is the main frame in the cutting cell protein, the structural protein’s main function is to maintain the integrity and continuity of the epithelial tissue. Study cytokeratin (CK) the conservatism of the high specificity and tissue differentiation, is closely related to the epithelial cell proliferation differentiation. Has now been confirmed source of cytokeratin 20 kinds of epithelial cells often positive expression. Vimentin as the main member of intermediate filament protein family, almost all expression in normal mesenchymal cells, to maintain cell integrity and against outside emergency injury plays an important role, and the flexibility of waveform proteins on cells is very important.Found in the pressure test of the tube, vimentin can provide the microtubules and actin did not elastic, so the waveform protein is responsible for maintaining the integrity of the cytoskeleton.In addition, in the absence of vimentin cell by a small needle, there will be a serious injury.In eliminate waveform protein gene of laboratory mice, although they have the function of normal, but suffer from loss of vimentin micro network waveform proteins were also found to be responsible for controlling the lysosome transfer derived by low density lipoprotein (LDL) cholesterol to etherification loci. When the transport is blocked, will accumulate less than usual lipoprotein in the cell. This relationship is need to rely on the screen in the middle of the cell of the first biochemical process, and also has a branch in the adrenal gland cells, is dependent on generated from LDL cholesterol ester.Traditional pituitary tumor cell culture mostly in the first place will change suspended growth of pituitary tumor cells into stick wall, for proliferation. But the 2D cultivate system is not very good simulation of tumor microenvironment. Bissel MJ’s team consider that 2D cultivation can make cells lose many signaling pathways, in the process of cultivation, as far as possible to reduce cell natural structure is essential to the intercellular signal exchange.In the body, solid tumors in 3D space can be infinite proliferation, and signal exchange activity between cells and extracellular matrix, cell growth polarity, spontaneously gathered together, and in the traditional culture system in vitro, cells are attached to a dish bottom, monolayer culture, cells and cells, cells and cell matrix signal exchange only from a plane, cell growth form non-polarity cells, cannot reflect the real situation in the body.Regard to the two problems of cultivating the pituitary adenoma,1. To remove fibroblasts in the cultivate system.2. Cultivate stable proliferation and maintain good function of pituitary adenoma cells. In order to improve human pituitary adenoma cultivating mode, we combined with domestic and foreign research experience . To overcome the existing cell lines the influence of different species, which can simulate the natural human body tumor traits better. To remove fibroblast in culture system as far as possible, and restore the natural structure, cell growth promoting intercellular signal exchange, to keep the good function of the cells in vitro environment, looking for a long-term, stable and efficient culture methods of pituitary adenoma for pituitary tumor related experiments such as:development of tumor drug targets, endocrine function recovery potential, to provide human pituitary adenoma cells as experimental material.After primary culture of pituitary adenomas, through the determination of CK and the expression of Vimentin, we can identified the cultured epithelial cells of the pituitary adenoma cells remain the characteristics of stable proliferation, and maintain the function.MethodsUse of hospital neurosurgery from November 2014 to June 2014 examination for fresh surgical specimens of 36 cases of pituitary adenoma, postoperative pathology confirmed for pituitary adenoma,8 cases, including growth hormone adenoma type prolactin,10 cases were adenoma, follicle-stimulating hormone type adenoma,8 cases,10 cases of functional adenoma.According to a report in the literature on culture system with growth time, the continuous proliferation of fibroblasts, repeatedly used differential adherent method after purification of pituitary adenoma cells suspended within 24 orifice plate, and with D-valin DMEM for culture. During culture, once the fibroblast was found, change culture vessels immediately, continue to develop. Eventually, fibroblasts in cultivate system be maximally eliminated, and keep the pituitary adenoma cells suspension growth characteristics, promote its signal exchange between cells. And we keep the pituitary adenoma suspension characteristics in the process of cultivation, on the one hand, can facilitate the separation of pituitary adenomas and fibroblasts, on the other hand, in a liquid medium environment, pituitary tumor cells have a tendency to natural aggregate, more conducive to signal exchange between cells. Cells by observing the microscopic morphology, cell immunofluorescence chemical staining and cell proliferation experiment to identify the primary cell.Result1. By the improved training method to create the pituitary tumor cell suspension in culture, have trend of gathering, bright and refraction, the original generation training batches success 28 cases (77.78%).Survive at least 15 days, the longest surviving 112 days, an average 41.81 days, stable and be able to extend the longest five generations and maintain good function.2. Proliferation experiments in vitro have shown that cells have good ability to multiplication and division.3. Via immunofluorescence chemical dyeing appraisal, epithelial cell marker cytokeratin (CK) is positive. And Vimentin, the mesenchymal origin cell marker is negative.4. Western Blot experiment shows epithelial cell marker cytokeratin positive. And Vimentin, the mesenchymal origin cell marker is negative.Conclusion1. Each type of pituitary adenoma cells are difficult to stick to wall, but suspended growth, and has a tendency to gather.2. Pituitary adenoma is suspended or half adherent growth, however the fibroblasts attached growth, using this feature, repeated on a regular basis, by applying the method of differential sticking wall change a dish, you can minimize the cultivation system of pituitary adenoma fibroblasts, achieve the goal of purification of pituitary adenoma cells.3. Pituitary adenomas were suspended growth naturally and has tendency of gathering and keep pituitary adenomas suspended growth can maintain signal transmission between pituitary tumor cells, also stable to extend and good function.Part Two The IL-6 Effect on Pituitary Adenoma of Pituitary DysfunctionBackgroundPituitary dysfunction is a kind of chronic disease causes [1]. endocrine pituitary dysfunction was different, occult disease, morbidity and mortality and hormone levels and hormone elevated or lack of related to the degree of pituitary dysfunction. The most common disease because of intracranial tumors, with sellar tumors (the most common for example, pituitary adenoma, craniopharyngioma), parasellar tumors (such as the second, parasellar meningioma, optic nerve glioma), again for intracranial metastatic tumors (such as breast cancer, brain metastasis, brain metastasis of lung cancer, melanoma brain metastases) [2]. generally increases due to compression of the pituitary tumor or pituitary disease, or because of the pressure in the saddle may lead to the increase of pituitary blood supply disorder, causing the pituitary dysfunction. But the progression of pituitary dysfunction disease onset of such situation is slow, unless the patient once tumor stroke, stroke, Pituitary endocrine function disorder disease in a few hours to days worse.The pituitary adenomas were the third intracranial tumors, accounnting 10%. domestic statistics the incidence rate is about 1/10 million, the annual incidence rate of female was 70/100 million,28/100 million men, the autopsy rate is about 20% 30%[3,4]. and prolactinoma is the most common pituitary adenoma can, accounting for all pituitary adenomas in 40%-60%[4]. of pituitary adenomas mainly harm the human body from the following aspects:1 pituitary hormone secretion caused by excessive metabolism and organ function damage in a series of 2; tumor oppression made low other pituitary hormone levels, caused by the dysfunction of corresponding target gland; 3 compression sellar structures, such as cross, optic nerve, cavernous sinus, cerebral artery, hypothalamus, third ventricle, even involving the frontal lobe, temporal lobe, brain stem, resulting in serious obstacles to the corresponding function [5]-For adenoma of endocrine function. It is generally believed that the patient’s clinical symptoms depend mainly on the endocrine function of tumor, such as prolactin cell adenoma of clinical symptoms to amenorrhea galactorrhea syndrome mainly. But the clinical findings a lot of patients with amenorrhea galactorrhea symptoms attending, and divided into endocrine examination also showed that patients with high serum prolactin levels, the final pathology confirmed non prolactin cell adenoma; or pathology of prolactinoma patients without amenorrhea galactorrhea syndrome.Interleukin6 (IL-6) not only involved in immune regulation and regulation role of endocrine.IL-6 can effect on the pituitary gland, placenta and other organs, regulating hormone secretion [6,7. In rat pituitary and IL-6 stimulates the secretion of follicle, resulting in prolactin (PRL) and growth hormone (GH) increased. In the human placenta and IL-6 stimulated chorionic gonadotropin release of pro, which in the structure and function of luteinizing hormone (LH) similar [6,7]The purpose of the researchAccording to the previous studies found that inflammatory factors in pituitary dysfunction plays an important role, we through the experiment explore effect and mechanism of inflammatory factor in pituitary endocrine disorders, inflammation of the pituitary tumor control may be beneficial for the treatment of endocrine disorders. A new target at the same time according to the target of IL-6 to treat possible future therapy for pituitary endocrine disorders.Experiment methodThrough the collection of the south hospital from November 2014 to June 2015, 25 cases of suspected pituitary adenoma accompanied by endocrine dysfunction patients plasma. This study by the hospital ethics committee agreed that the subjects signed informed consent.We make use of the liquid phase of suspension biochip detection method and contrast pituitary adenoma with pituitary dysfunction patients and in the peripheral blood of 8 kinds of common inflammatory factor expression. We use expression of IL-6 increased significantly at the dose of many stimulating human prolactinoma cells and by ELISA analysis detection of IL-6 and tumor cell hormone secretion, and explore its potential mechanism.Result1. IL-6 in pituitary adenoma with significantly increased in patients with pituitary dysfunction in peripheral bloodThe peripheral blood samples of 25 cases of pituitary adenomas in peripheral blood samples and 3 cases of normal healthy people will be included in the test, the pituitary adenoma with endocrine dysfunction and peripheral blood inflammatory cytokines expression level significantly increased IL-6, and there was significant difference.2. The concentration of IL-6 and prolactinoma related hormone secretion Different concentrations of rhIL-6 intervention lactation prolactinoma cells 48 hours after that of prolactinoma cell prolactin secretion has obvious role in promoting, and compared to the control group, prolactin secretion increased, at 500 units/ml, P< 0.05, with the increase of drug concentration, promote the effect of prolactin secretion is stronger, a dose dependent effect.3. the effect of dopamine receptor agonists can attenuate IL-6 induced prolactin secretionBromocriptine combined with IL-6 intervention lactation hormone adenoma cells after 48 hours, IL-6 induced lactation prime adenoma cells lactation hormone secretion inhibition, and compared to the control group, prolactin secretion increased, compared with the group, IL-6 prolactin secretion is reduced.Conclusion1. IL-6 may be one of the causes of the disorder of the endocrine function of pituitary adenomaThe comparison of 25 cases of pituitary adenomas in peripheral blood samples and 3 cases of normal healthy people blood samples, we found IL-6 showed significantly higher expression, and there was significant difference. And the effect of rhIL-6 on lactation hormone pituitary adenoma, found a corresponding increase in with the increase of the concentration of rhIL-6 and prolactin secretion, and reach 500U/mL concentration rhIL-6 exist significant difference.2. IL-6 induced prolactinoma hormone secretion can be dopamine receptor agonists inhibitBy added to the culture system of bromocriptine we found in the rhIL-6, stimulate prolactin cell adenoma of the secretion of prolactin effect is reduced, indicating that bromocriptine in dopamine receptor and agitated, can also reduce IL-6 on prolactin cell adenoma of the stimulus, thereby reducing lactation element release, so as to achieve the treatment of prolactinomas purpose. At the same time, the effect of IL-6 on prolactin release of dopamine receptor dependent. |
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