The Improved Culture Method Of Craniopharyngioma Primary Cell And The Establishment Of Immortalized Cell Line

Background and ObjectiveIn 1930s, the father of modern neurosurgery, the American Association of Neurological Surgeons Harvey Williams Cushing (1869.04.08-1939.10.07) professor once said:"craniopharyngioma is one of the most depressing diseases." In China, famous neurosurgeon professor Qi Songtao said:"craniopharyngioma is the most challenging diseases for neurosurgeons, surgical treatment and postoperative management of the most embodies the neurosurgeon level." Although more than 100 years, the origin of craniopharyngioma understanding and surgery made progress, but the lack of a stable tumor cell lines, namely the lack of appropriate research platform, basic research is still craniopharyngioma was weak.The late 19th century, a number of pathologists noted that the growth of a rare type of epithelial tumors in sellar. In 1904, the Austrian professor Erdheim first detailed description of the histological features of the tumor, the tumor occurrence and proposed related to the Eustachian tube embryo, and as a sellar tumors separate class of diseases are described. Then in 1932, the US Department of Neurosurgery Professor Harvey Williams Cushing in the book is the first to be named "craniopharyngioma" and detailed description of them, and thus a standardized name, still in use.Craniopharyngioma is the most common tumor in children saddle area, formed from ectodermal remnants cranial pharyngeal tube epithelial cells developed a common residual embryonic tissue tumors, is also the most common congenital intracranial tumor. Tumorigenesis Currently there are two main controversial theory. The first one is congenital remnants of embryonic epithelial cell theory:the second week of embryonic development, the original roof of the mouth begin projecting upward, gradually developed into an elongated blind sac that Mubarak bursa (Rathkes Pouch), located at the front of the spinal cord. Later some of the former fossa appears at the bottom funnel downward projection, the two gradually close, and finally the development of the pituitary. Rathke’s bag and the original oral tapering part connected to a bureaucratic, called cranial pharyngeal tube. Rathke’s bags in the first eight weeks after the rapid proliferation of epidermal structure is formed by a simple adenohypophysis (anterior pituitary, tuberosity), the funnel is formed neurohypophysis (posterior pituitary). Normal pituitary tubercle near the funnel there are remnants of epithelial cells, most scholars believe that the origin of craniopharyngioma in these remaining cells. The second epithelial cell metaplasia theory:Many scholars believe that the epithelial cells of the pituitary tubercle pituitary cell metaplasia of the product, rather than the residual embryonic cells. The current interpretation of the origin of tumor there are still many points of supplementary evidence, it is controversial.In 2007, WHO pathological type of central nervous system tumors were divided into enamel type craniopharyngioma (adamantinomatous craniopharyngioma ACP) and squamous papillary craniopharyngioma (papillary craniopharyngioma PCP) two types, the former is about 90%. Although WHO pathological grade both belong to Class I, but showing a clinically "benign pathology, malignant biological behavior" performance, in particular, occur in the enamel type craniopharyngioma severe inflammation in the hypothalamus and brain tissue adhesions violations. Enamel type craniopharyngioma tumor staining substance paraffin outer periphery of simple columnar epithelial cells "fence-like" arrangement, which can be seen the inside of a large "Star Network loose" cells, tumor infiltration of inflammatory cells seen in the center, "turbine-like" cells, dentin body, wet keratosis, calcification, necrosis of apoptotic cells, cholesterol crystals and other structures. And hypothalamic tumor at the junction of the outer brain tumor tissue and tumor was gliosis was "finger" violation of brain tissue. Squamous papillary craniopharyngioma pathological HE staining tumors were regularly arranged in papillary squamous cell and inflammatory cell infiltration occurs mainly in the tumor stroma, light than the enamel type tumor and surrounding brain tissue boundaries clear.Surgical treatment is the preferred method of treatment of craniopharyngioma, and the only cure for the disease. With the development and application of imaging and microsurgical instruments, craniopharyngioma origin and type of surgical approach to understanding the concept of innovation, the first positive surgical resection of tumor gradually recognized by the vast majority of neurosurgeons. However, the importance and the adjacent structures deep in craniopharyngioma position, the outer periphery of the tumor and inflammatory adhesion traction, brain tissue invasion and calcification, so that the rate of surgical resection, tumor recurrence rate and postoperative quality of life clinical treatment processes craniopharyngioma still facing problems.Stable tumor cell lines lacking, hindering the development of basic research craniopharyngioma, makes the most of now stands at histological level to explore the development of tumors, inflammation, endocrine, calcification, recurrence. Now that the enamel type craniopharyngioma mainly in the pathogenesis of early embryonic development, Rathkes capsule remaining cells encoding β-catenin in CTNNB1 exon3 mutated such that β-catenin degradation complex and can not be combined resulting in its accumulation in the cytoplasm and into the nucleus, leading to tumorigenesis. In 2014, German neuropathologist Professor Holsken enamel type craniopharyngioma first confirmed on histopathology "turbine-like" cells co-expressing β-catenin into the nucleus of the cancer stem cell marker CD133, CD44, and this type of cell subsets named craniopharyngioma tumor stem-like cells (craniopharyngioma stem-like cells, CSLCs). Professor Holsken confirmed in its text CSLCs exist only in the enamel type craniopharyngioma, this tumor development is closely related to tumor recurrence.Human-derived cell lines craniopharyngioma craniopharyngioma biology is the study of the characteristics of one of the most important means for the development of cancer, the cause of pathological mechanisms, clinical exploration and evaluation. Craniopharyngioma belong to epithelial cells derived from benign tumors, in vitro and establish a stable cell line is particularly hard, indirectly led to the in-depth study craniopharyngioma little cytological level, to carry out a combination of basic and clinical research Translational Medicine there are serious problems. As early as 1997, the German Professor Honegger first reported successfully cultured enamel type craniopharyngioma cells in cell culture demonstrated short-term progestin in combination with progesterone receptor surface of tumor cells and promote tumor value; then in 2005, the German professor Ulfarsson trypsin digestion cultivate enamel type craniopharyngioma cells, and confirmed that insulin growth factor Ⅰ (insulin-like growth factor 1 IGF-1) can promote the value of their tumor cells; in 2010 a German professor Holsken optimized keratinocytes culture medium enamel type cranial pharyngeal tube cells, and confirmed that overexpression of migration CNTTB1 gene can promote tumor. In China, in 2010, Changzheng Hospital, Second Military Medical University, Professor Jiang Rong was the first time a hybrid trypsin and collagenase digestion and cultured cells craniopharyngioma, and bleomycin demonstrated in vitro to inhibit the proliferation of tumor cells; in 2011, West China Hospital, Sichuan University Xu Jianguo and Professor Rollins reported separately cultured primary cells craniopharyngioma, the former confirmed growth hormone promotes the proliferation of the tumor, and tamoxifen inhibit tumor growth, which confirmed that retinoic acid can induce primary craniopharyngioma cells apoptosis. In 2013, Southern Medical University, Professor Greenfield paint continue to improve primary cell culture methods, and explore more suitable craniopharyngioma cell growth medium in vitro can pass 6-9 generations. The same year, West China Hospital, Professor Yu Chao Professor Holsken learn using glial cell culture commercialization of continuous improvement culture craniopharyngioma cells. In 2015, Professor Tian Zengmin Navy General Hospital reported trypsin digestion differential adherent method, craniopharyngioma successfully cultured cells. Above craniopharyngioma cell culture methods reported in the literature at home and abroad, think of themselves, we found many problems:Most craniopharyngioma cell culture literature is limited to the first generation; some reported craniopharyngioma there are a lot fibroblast cells pollution; some reported the cultured cells are actually non craniopharyngioma cells, but tumor-associated fibroblasts; in addition, each cell morphology craniopharyngioma reported different culture, namely cells corresponding changes in morphology and stability occurred.The so-called immortalized (cellimmortalization), refers to the in vitro culture of primary cells through spontaneous or affected by various factors in order to escape from the outside world, to escape from the proliferation of bad crisis, so as to have the ability to process an unlimited steady proliferation. I. primary cultured human primary cultured cells spontaneous immortalization quite rare chance, therefore, efforts by gene transfection technology exogenous immortalizing gene, such as viruses, proto-oncogenes and tumor suppressor gene mutants, etc. in the generation of cells, promote immortalized occur, in order to establish a stable immortalized cell lines (immortalizedcell line), to reach the cells were immortalized and primary cultured cells maintain biological properties. In recent years, the study found re-activating cells immortalized with telomerase are closely linked. Thus, exogenous hTERT telomerase gene directly activates intracellular so that cells are obtained immortalized by viral transfer methods. Currently extensive literature studies have shown that the virus transferred by exogenous human telomerase reverse transcriptase (hTERT) gene has established a considerable plurality of proliferation and ecology stable cell lines, such as endothelial cells, neural progenitor cells, meningioma, signet ring cell carcinoma, prostate epithelial cells, retinal pigment epithelial cells, mammary epithelial cells and bone marrow stromal stem cells, wherein the cells reactivate telomerase activity, maintaining the original morphology of cultured cells without malignant transformation.In summary, the optimization of primary cell culture craniopharyngioma, especially the activity of cell biology, morphology and stable proliferation is essential; in-depth study of these turbines having characteristics of stem cells transfected cell that craniopharyngioma tumor stem cells like cells (CSLCs), to explain the occurrence of the craniopharyngioma enamel type, as well as the development of brain tissue and tumor invasion, tumor calcification is particularly important; in good primary cells, based on the establishment of a stable immortalized cell craniopharyngioma Department of craniopharyngioma biological characteristics, the significance of the development of cancer, etiology pathogenesis, clinical treatment and other major.Content and Methods1.The improved culture method of craniopharyngioma primary cell and identificationCollect Nanfang Hospital of Neurosurgery January 2015 to December 2015,85 cases of craniopharyngioma surgical specimens, all samples were diagnosed by the hospital pathology craniopharyngioma. Wherein ameloblastoma type craniopharyngioma 66 cases,19 cases of squamous skin type craniopharyngioma; children accounted for 46 cases of adults accounted for 39 cases.85 cases of specimens are subjected to conventional dehydration embedded in paraffin. There are 49 cases in which the tumor tissue due to more substantive component, the methodology was modified craniopharyngioma cell culture using an inverted microscope photographs recording its primary cell and passaged, the cultured cells before and after repeated contrast, continuous optimization culture conditions. Select the cultured cells were passaged cells were identified by immunofluorescence, indicators include the Pan-CK, VIM, a-SAM, to further clarify craniopharyngioma epithelial cells and fibroblasts, namely tumor-associated fibroblasts. Experimental cell biology cells selection state after a good passage, CCK-8 experiments including cell proliferation, cell monoclonal experiments, cell cycle, apoptosis.2. Adamantinomatous craniopharyngioma swollen cultured stem-like cells and multipotential differentiationIn front of the 85 cases of craniopharyngioma paraffin-embedded blocks, conventional serial sections of immunohistochemical staining, indicators, including CD 133, CD44, P-catenin and HE staining. Under the upright microscope photograph observation ameloblastoma and papillary type of CD 133, CD44, β-catenin expression distribution.Craniopharyngioma front purified culture cells, after passage one containing DMEM/F12 (1:1), LIF, B12, EGF, bFGF and other factors add cell suspension culture medium was centrifuged medium was changed every 2-3 days, different time periods observed and photographed tumor formation rate and the size of the ball changes inverted microscope. After 2 weeks of culture, the cells were detected by immunofluorescence were made Hsien craniopharyngioma tumor stem-like cells (CSLCs) ordinary craniopharyngioma cells CD133, CD44 and expression of β-catenin, Western-blot and RT-PCR continues to detect two or more kinds of cells CD133, CD44, and β-catenin expression. Until the cells covered 70%, the use of osteogenic inducer (medium containing 10 mmol/L, β-glycerophosphate,50 mg/L of vitamin C,10 nmol/L dexamethasone) more than two kinds of cell culture induce and maintain more than 21 days. After induction, the end of Alizarin Red S detected more than two cell osteogenic differentiation.3. Preliminary exploration craniopharyngioma immortalized cell linesEarly expression lentivirus construct better and explore viral transfection MOI value. Choose good condition craniopharyngioma primary cells, adherent cells covered 60% -70%, the medium was changed after lentivirus transfected expression of hTERT gene transfection within 24 hours to wash viral transfection solution. Transfection 48 hours,72 hours,96 hours, respectively, to take pictures, to detect the expression of green fluorescent cells and strength. Finally, RT-PCR was used to detect gene transfection mesh cells, load cells transfected, untransfected control cells hTERT mRNA levels change.4. Statistical methodSPSS 170 statistical software using the above test results the appropriate statistical analysis. Measurement data with X ± SD between the two groups were compared using two independent samples t-test was used to compare among groups of One-Way ANOVA for comparison. P<0.05 indicates a statistically significant difference.Results1. Craniopharyngioma improved primary culture and identificationModified cranial pharyngeal tube tumor cells in primary culture and viable cells, which showed a stable growth passage,4 to 9 generations; two pathological types of craniopharyngeal duct tumor cell culture morphology had no significant difference, with a pathological types of craniopharyngeal duct tumor cells between different individual morphology are small differences, adherent cell morphology was "cobblestone" arrangement; cell culture, purification, craniopharyngeal duct tumor cells by immunofluorescence for Pan cytokeratin (Pan-CK) positive, vim and negative alpha also negative, cranial pharyngeal duct tumor related to fiber cell immune fluorescence pan cytokeratin (Pan-CK) negative, VIM was positive, alpha also positive. The cell growth curve was "S" type, and the growth curve was different between different cells. Most of the 3 generations of the primary cell of the primary cell of the cranial pharyngeal canal were in the S phase.2. Adamantinomatous craniopharyngioma culture and identification of the glaze type craniopharyngioma tumor-like stem cellsImmunohistochemistry showed that adamantimous craniopharyngioma tissue tumor cells co expressing turbo beta-catenin into nucleus and tumor stem cell markers CD133, CD44, and other parts of the tumor cells and expression of CD133, CD44 and beta -catenin nuclear scales, craniopharyngiomas and expression of CD 133, CD44 and beta -catenin into the nucleus; 2 weeks after the tumor stem cell suspension culture method of adamantimous ball, shows a large number of the size of the tumor stem cell spheres, and a scaly skin type after 2 weeks of culture, there is no stem cell tumor sphere formation (P=0.006); immunofluorescence showed tumor ball adherent co expression of beta -catenin into nucleus and tumor stem cells markers CD133, CD44, common adamantimous craniopharyngioma cells expressing only-catenin beta cell membrane, tumor stem cell marker CD133, CD44 negative; Western-blot showed tumor stem cell spheres and normal cells of stem cell markers CD133, CD44 and beta-catenin Protein level expression has a significant difference (P< 0.001, P< 0.001; P= 0.024); qt-pcr detection stem cell tumor ball and ordinary CD133, CD44 and beta catenin mRNA expression level has a significant difference (P< 0.001, P< 0.001; P= 0.001); osteogenic induction medium for 21 days, cranial pharyngeal tube tumor stem cell like cells compared with ordinary craniopharyngeal duct tumor cells, alizarin red staining was strongly positive.3. Preliminary exploration craniopharyngioma immortalized cell linesGood condition craniopharyngioma primary cells transfected by lentivirus hTERT 48 hours,72 hours,96 hours after the fluorescence expression were 13%,54%,89%, then the current turn seven generations has been passed, the state is still good. Transfection of target gene mRNA levels of hTERT was significantly higher than the no-load transfected cells, untransfected control cells.ConclusionCraniopharyngioma improved primary culture, its shape and activity was significantly superior to other methods currently reported in the literature for the future in-depth fundamental research to provide a better platform, but also to establish a stable immortalized cell lines and premise basis; in first cellular level confirmed the presence of enamel type craniopharyngioma CSLCs, cancer stem cells have multiple differentiation potential, in order to explain the diversity of pathological enamel type craniopharyngioma do the preliminary work; in addition to our next CSLCs tumor inflammation release of brain tissue invasion, resistance to chemotherapy put the foundation; craniopharyngioma preliminary exploration of immortalized cell lines, the progress of these experiments will lay a solid foundation for further study late craniopharyngioma.