| Neurofibromatosis typeⅡ, also known as central neurofibromatosis, is a autosomal dominant genetic disease, with an incidence rate of 2.50/100000. The pathogenic gene responsible for the disease is NF2, the tumor suppressor gene, locating on chromosome 22q12.2,120kb in length approximately. The NF2 gene encodes Merlin protein, its mutation results in inactivation deficiency of Merlin protein, causing uncontrolled cell growth leading to tumor formation. The disease manifests as bilateral vestibular schwannomas, multiple meningiomas and other tumors. Bilateral vestibular schwannomas are the main clinical feature that often appears as tinnitus, hearing loss, vertigo and balance dysfunction. Neurofibromatosis typeⅡhas a serious impact on the life quality and life span of patients. To explore more effective management for the disease, studies on targeted therapy at cellular level, drug screening and personalized sequencing for NF2 gene mutation are helpful. It has been shown already by researches that schwannoma might arise from the the schwann cells. The extraction and purification of neoplastic schwann cells in this study will establish in vitro experimental platform, based upon tumor specimen for patients with neurofibromatosis typeⅡ, which would shed light on the further study aimed at improving the medical management of neurofibromatosis typeⅡ.About 50% of patients with neurofibromatosis typeⅡare inherited, another 50% are the result of new de novo mutations. A variety of mutation types have been detected in NF2 gene. It is very valuable to find individual differences at genetic level in neurofibromatosis typeⅡpopulation by gene sequencing. This study will develop a method of molecular genetic analysis for NF2 gene. By comparing the individual gene mutation type, we would find novel mutation types and determine whether specific mutations are associated with respective disease types. By comparing genotypes of neurofibromatosis typeⅡbetween patients and their offspring, we would predict the possibility of the offspring who were the potential neurofibromatosis typeⅡpatients. The genetic counseling, long-term follow-up and comprehensive management would be schemed for the patients and their families.This study includes two parts as follows:Part I:The purpose of this part is to establish the method of extraction and purification of neoplastic Schwann cells in vitro, obtain pure and sufficient cells cultured and improve successful rate of primary cell culture in neurofibromatosis typeⅡ. Ten fresh tissue specimens of Schwannoma were used as experimental material, which were collected from patients with neurofibromatosis typeⅡ. Neoplastic schwann cells were extracted after I collagenase digestion. The purification step was performed through rapid digestion with low concentration trypsin, differential detachment and clone screening. Cell morphology was observed under inverted microscope, and cultured neoplastic schwann cells were identified immunocytochemically by S-100 protein staining. The results showed that neoplastic Schwann cells were successfully obtained in 5 cases of 10 tumor tissue specimens. A large number of bipolar spindle or triangular-shaped cells could be seen on the third day of primary culture after trypsin digestion with low concentrations and differential detachment purification; When cultured for 3 generations,4 to 5 cell clones appeared under the inverted microscope with S-100 protein positive staining. The purity of neoplastic schwann cells in this study was larger than 95%. The cell viability was fine and the cells were able to be passaged stably in the subculture.PartⅡ:The purpose of the second part is to establish molecular genetic analysis method applicable clinically for molecular genetic diagnosis in patients with neurofibromatosis typeⅡand their offspring, and further to guide genetic counseling, patient's condition monitoring and follow-up as well as the clinical intervention. Genomic DNA was extracted from neoplastic schwann cells and blood in two patients and their offspring who agreed to accept gene sequencing. The NF2 gene was sequenced (E1～15 and E17 exons and adjacent introns). Based on the patient's clinical manifestation and symptoms, reasonable plans for clinical interventions and follow-up were develop. According to the implication of NF2 gene sequencing, genetic counseling was given to the neurofibromatosis typeⅡfamily, and the potential neurofibromatosis typeⅡpatients in offspring were followed-up in long term. Schwannoma tissue and genomic DNA bank was established initially in our department. The totally same NF2 gene mutations were detected in genomic DNA extracted both from neoplastic Schwann cells and blood cells in the single patient in this study. By comparing the genotypes between the patients and their offspring, the consistent NF2 gene mutations were found between a female patient and her daughter aged 3, but not completely consistent gene mutations were detected between another female patient and her son aged 15. All of the mutations in NF2 gene were located in the control region near the exons. Conclusion:It is an effective method to obtain highly pure and viable neoplasitc Schwann cells without the mixture of fibroblasts by primary cell culture in vitro through rapid digestion with low concentration trypsin, differential detachment and clone screening. These neoplastic Schwann cells could be applied in the further studies on pathogenesis and treatment for neurofibromatosis typeⅡ.The tumor tissue and genomic DNA bank of neurofibromatosis typeⅡcould supply the bio-resource for genetic molecular testing and treatment studies. Molecular genetic analysis would be applied in clinical practice guidance and risk prediction for neurofibromatosis typeⅡ. For high risk individuals potentially suffered from neurofibromatosis typeⅡ, follow-up plan should be made and early diagnosis and early treatment should be ensured. The condition monitoring and long term follow-up, personalized clinical intervention are needed to improve the quality of life and prolong survival. |
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