Mechanism Research Of BMP2-induced Osteogenic Pathway In The Calcification Of Craniopharyngioma And The Establishment Of Human Craniopharyngioma Xenografts In Chick Chorioallantoic Membrane

BackgroudCraniopharyngioma is the most common epithelial tumor of central nervous system, which grow along the along the cranial pharyngeal tube. This tumor accounts for4.7%-6.5%of all intracranial tumors and constitutes6%-13%and60%of all children intracranial tumors and sellar region in children, respectively. Craniopharyngmioa was associated with the hypophyseal-pharyngeal duct of embryo and described as aindividual sellar tumor by Erdheim a Austrian. The name craniopharyngioma was changed among the name of Ratheke’s tumor, hypophyseal tumor, cranial-pharynx duct tumor and ameloblastoma. The name craniopharyngioma was first used by Frazier and Alpers in1931. Since the description by Cushing using the word craniopharyngioma in his treatiseintracranialtumour, this name was being continuous to be used. The view in the pathological features and biological characteristics of cranipharyngmioa is still controversial due to the lack of the tumor research platform and technical conditions, although the find research on this tumor by scientist in the world have been made for more than100years.Craniopharyngiomas are histologically classified into two types: adamantinomatous craniopharyngioma and squamous papillary craniopharyngioma, which are deemed to be a benign tumor by WHO. ACPs are often occur in child, featuring in the calcification, inflammation and cysticdegeneration, whereassquamous papillary craniopharyngioma are mostly occurs in adult by a solid tumor. For the various growing form and its adjacent to some important structures(pituitary stalk, opticnerve, opticchiasma and hypothalamus et al.) organization around craniopharyngioma are tend to be injured when totally remove the tumor in the surgery. This may induce the serious complications and even death. On the other hand, the subtotal ectomy almost means the happen of tumor recurrence. Therefore, it is a great challenge for neurosurgeons to total remove the tumor and to avoid the injury of structures around simultaneouslyAmong risk factors during the treatment of craniopharyngioma, calcification is a more important factor. Calcifications are common in cranipharyngioma with a calcification rate of approximately60%for the adults and more than85%for children. Clinical data indicate that, craniopharyngioma calcification often associated with pituitary stalk, optic chiasm, internal carotid artery, and third ventricle structure closely, which causes great difficulties for completely stripping during surgery. Calcification of craniopharyngioma is composed of hydroxyapatite combined with a variety of fibers. Division and resection of the calcification mass was difficult for its toughness and the overall remove easily cause injurys of surrounding structures and the serious consequencesThe clinicopathological data show that calcifications are often found in adamantinomatous craniopharyngioma but not in squamous papillary craniopharyngioma. Histologically, adamantinomatous craniopharyngioma resembles odontogenic epithelial tumors.The the enamel proteins expressed exclusively in the tooth have also been found in adamantinomatous craniopharyngioma but not in squamous papillary craniopharyngioma. Considering the differentiation of hypophyseal-duct during its development process, some researchers believe thatadamantinomatous craniopharyngiomaoriginates from the odontogenic rests associated with remnants of Rathke’s pouch and that squamous papillary craniopharyngioma develops from the buccal mucosa rests, although these two typs are both developed from the remnant of the embryo hypophyseal-oropharyngeal duct. It is reasonable to consider that adamantinomatous craniopharyngioma is a benign tumor with characteristics of epithelial odontogenic tumor.Our previous study found that OPN was elevated in adamantinomatous craniopharyngiomaespecially in the calcified samples and weak in squamous papillary craniopharyngioma. As a secretory protein, OPN show strong affinity to the calcium salt for its special structure. During the formation of bone and tooth, OPN acts as a cross-linking factor and is localised at matrix-matrix, matrix-mineral, and matrix-cell interfaces and between collagen fibrils of fully matured hard tissue, and could be synthetized by osteoblast and osteoclast. Thus OPN could be deemed as an important factor in the anabolism and catabolism of bone.Bonemorphogeneticprotein is a growth factor withosteogenic inductiveness, which belongs to the transforminggrowthfactor-βsuper family. BMPs are effective in the cell proliferation, differentiation and invation, except for the induction of bone formation. BMPs were attracted from the bone and capable of the induction of new bone formation, which make them as a material in aiding the healing of fracture for more than10years. BMPs could gather and stimulates the differentiation of mesenchymal stem cells (MSCs) into an osteoblast lineage, which lead to the secretion of osteogenic proteins including OCN, OPN, collagen and ALP, calcium deposit and bone formation.BMP2also function on the formation of tooth by inducing the differentiation of ameloblasts and odontoblasts. Kumamoto and his colleagues found that BMP2, BMP4and BMP7were expressed in the odontogenicameloblastomas and tooth tissue and consider BMPs were important in the differentiation of ameloblasts and the formation of tooth. In addition, Bmp2also plays a role in pathological conditions, such as tumour calcification and vascularsmoothmuscle calcification. The smooth muscle cells and fibroblasts could be stimulated by BMP2and differentiate into aosteogenic lineage.Based on the expression of BMP in the odontogenic tissues and the important roles of OPN in the calcification of craniopharyngioma, we detected the expression of BMP2a typical member of BMPs and two osteogenic transcription factor RUNX2 and OSTERIX in caniopharyngioma andanalyse their expression patterns to explore the mechanism of craniopharyngioma calcification. We used the human recombinant BMP2to stimulate craniopharyngioma cells and detect the expression of key factors like RUNX2, OPN, and ALP, to preliminary confirm the induction of BMP2osteogenic path way in craniopharyngioma. We also test the calcium deposit of craniopharyngioma by using thealizarin red staining. We establish the craniopharyngioma xenografts in chick chorioallantoic membrane a hypoallergeniccarrier to explore the experiment platform for the further study.Chapter I Section I The expression of BMP2, RUNX2and OSTERIX in craniopharyngiomaObjectiveTo test the expression of BMP2, RUNX2and OSTERIX in craniopharyngioma and explore the relationship between these factors and the calcification of craniopharyngioma.MethodCollect the surgical specimens of craniopharyngioma, and retrospective analyse the relationship between expression of osteogenic factors (BMP2, RUNX2and OSTERIX) and the pathological type and calcification degree and the relationship among the proteins as well; Use the H&E staining to detect the the calcification lesionsother histological structures and analyse relationship between the expression pattern and the formation of calcification.ResultH&E staining:In some sample, the whorl-like array cell, wet keratin and calcification lesion coexist in a field. The calcification lesion seems to arise from its wet keratin. From some ossifying adamantinomatous craniopharyngioma, osteoidtissue with bone lacunae was observed. There is no calcification in squamous papillary craniopharyngiomaImmunohistochemical study:Of the77adamantinomatous craniopharyngioma samples,70.1%(54out of77),55.8%(43out of77), and50.6%(39out of77) demonstrated moderate and strongly positive expression (>10%positive cell) of Bmp2, Runx2and OSTERIX, respectively (Table2). In the12squamous papillary craniopharyngioma samples, there is no case showed moderate and strongly positive expression(>10%positive cell) for all osteogenic proteins. Bmp2displayed cytoplasmic and membranous expression while Runx2and OSTERIX showed nuclear and/or cytoplasmic staining. The pattern of staining of these three osteogenic factors was identical in the whorl-like array cells and stellate reticulum and staining was weak in the peripheral palisades. Remarkably, the expression of Runx2and OSTERIX tended to locate to the calcification-related epithelia including the whorl-like array cells and cells in/around the wet keratins and calcification lesions. All three osteogenic proteins showed no/little immunoreactivity in squamous papillary craniopharyngioma samplesThe expressions of Bmp2, Runx2and OSTERIX differed significantly between the adamantinomatous craniopharyngioma groups separated based on degree of calcification (P values<0.001, for all) and correlated positively with the degree of calcification in adamantinomatous craniopharyngioma (P values<0.001, for all). The levels of these three proteins in calcified adamantinomatous craniopharyngioma (those samples scored as+/++/+++) were higher than those in squamous papillary craniopharyngioma (P values<0.001for all) whereas there was no significant difference between SPCP and ACP (-)(Bmp2, Runx2, and OSTERIX, P=0.392,0.517,0.212, respectively). Moreover, there is a positive correlation between the expression of Runx2, OSTERIX and Bmp2in adamantinomatous craniopharyngioma (P values<0.001for all).Western-blot results indicated that the level of Bmp2, Runx2, and OSTERIX increased as calcification progressed in adamantinomatous craniopharyngioma(Figure6). There is no significant difference between the SPCP and ACP (-) groups for any of the three proteins (Bmp2, Runx2, and OSTERIX P=0.551,0.609,0.295, respectively, Figure6). The above WB results were consistent with the immunohistochemical findings.Conclusion BMP2and two transcriptional factors RUNX2and OSTERIX play important roles in the calcification of craniopharyngioma, BMP2might be induce the differentiation of craniopharyngioma into an osteogenic lineage and the calcium deposit in craniopharyngioma. Chapter Ⅰ Section Ⅱ The osteogenic differentiation of cranopharyngiomawas induced by Human Recombinant BMP2ObjectiveTo explore whether the ectogenichuman recombinant BMP2could induce the Osteogenic differentiation and the calcium deposit in craniopharyngioma cellsMethodUse the human recombinant BMP2to stimulate the craniopharyngioma and detect the gene expression of OPN. Test the protein expression of ALP, OPN, and RUNX2by using western-blot after the induction of BMP2in craniophayngioma and compare the expression level when NOGGINa BMP2antagonist was used. Perform the Alizarin red staining to observe the calcium deposit of craniopharyngioma.ResultQ-PCR:The mRNA expressions of OPN were measured using real-time PCR after10days culture. Our result showed that the expression of OPN could be slightly induced at the concentration of BMP2below50ng/ml, and markedly induced (7.6,4.9,8.7,5folds for4patients, respectively) at200ng/ml, when compared with the control groups.Western-blot:OPN and RUNX2were elevated by BMP2(P<0.001for both) while ALP shows no change when compared with the group CON(P=0.175). The expression level of these three factors in group BMP2+NOGGIN was lower than in group BMP2(ALP, OPN and RUNX2, P<0.001,=0.002and<0.001, respectively).Alizarin red staining:After14days of induction, the distinct calcium deposit were observed.The calcium deposit of group BMP2+NOGGIN was lower than the group BM2. There is no calcium deposit founded in the group CON.ConclusionCraniopharyngioma could be induced by recombinant BMP2and differentiate into a osteogenic lineage; By the stimulation of BMP2, the downstream factors in this osteogenic path way were elevated, which could be weaken by NOGGIN. This results confirm that BMP2pathway is the important approach for the craniopharyngioma calcification. Chapter Ⅱ The establishment of human craniopharyngioma xenografts in chick chorioallantoic membraneObjectiveTo establish the xenotransplanted tumor model in chick chorioallantoic membrane (CAM) and detect the angiogenesis ability, Microvesseldensity (MVD) and cell proliferation of the xenograft.MethodCraniopharyngioma tissues from operation were transplanted on the CAM. CAM angiogenesis assay was performed, and the MVD and PCNA was evaluated using immunohistochemistry.ResultThe tumor formation rate for adamantinomatous craniopharyngioma (ACP) and squamous papillary craniopharyngioma (SPCP) was47.14%43.33%.There is no significant difference between these two rate (P=0.726). The CAM angiogenesis assay, MVD and expression PCNA of adamantinomatous craniopharyngioma were higher than squamous papillary craniopharyngioma. The expression of PCNA was positively correlate with MVD and CAM assay score in CP(r=0.648, P<0.001, r=0.490, P=0.001, respectively).ConclusionAnimal model for human craniopharyngioma can be established in the CAM. The CAM angiogenesis of the xenograft can be evaluated and the craniopharyngioma xenograft of CAM process a new blood circulation and cell proliferation ability.