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Primary Schwannoma Cell Culture and Personalized Study of NF2 Gene Mutation in Neurofibromatosis Type Ⅱ

Author: WangWei
Tutor: YangXueJun
School: Tianjin Medical University
Course: Surgery
Keywords: Ⅱ neurofibromatosis type Schwann cells Primary cultured S-100 NF2 gene Molecular genetic testing Clinical intervention
CLC: R739.41
Type: Master's thesis
Year: 2011
Downloads: 33
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Abstract

The type Ⅱ neurofibromatosis, also known as the central nervous fibromatosis, is an autosomal dominant genetic disease, the incidence rate is about 2.50/10 pathogenic the responsibilities gene NF2 tumor suppressor gene, located on chromosome 22q12.2 The total length of approximately 120kb, Merlin protein encoded product. NF2 gene mutations cause the Merlin protein deficient inactivation induce uncontrolled growth leading to tumor formation. The the type Ⅱ neurofibromatosis mainly bilateral vestibular nerve Schwann cell tumors and multiple meningiomas. The bilateral vestibular Schwann cell tumor is the typical clinical features, usually manifested tinnitus, hearing loss, vertigo and balance disorders, and gradually lead to hearing loss and even loss of a serious impact on the patient's quality of life and life expectancy. In order to find a more effective treatment for type Ⅱ neurofibromatosis, targeted therapy of the disease from the cellular level, drug screening and NF2 gene mutations individualized study is particularly important. Studies have shown that neoplastic Schwann cells is likely to be of Schwann cell tumor-derived cells. This study by the type Ⅱ neurofibromatosis tissue extracted and purified tumor cells for further research to improve the medical treatment of neurofibromatosis type Ⅱ in vitro experiments platforms. Approximately 50% of patients with type Ⅱ neurofibromatosis clear family genetic history, of which 50% is caused by the new hair gene mutation. NF2 gene mutations in the form of a variety of individualized study of the disease at the genetic level, can more clearly understand the individual differences of the disease NF2 gene mutation and phenotype and found that the mutant form of the disease gene; NF2 gene comparison of patients and their children, whether the incidence in the future to predict patients' children is important. The present study intends to establish the type Ⅱ neurofibromatosis clinically applicable methods of molecular genetic analysis, molecular genetic diagnosis of the type Ⅱ neurofibromatosis patients and their descendants, in order to guide the the type Ⅱ neurofibromatosis family genetic counseling and individualized condition monitoring, follow-up and clinical interventions. This study is divided into two parts: Part 1: the establishment of the the type Ⅱ neurofibromatosis tumor extracted and purified Schwann cells in vitro, in order to obtain a sufficient quantity and purity of the cells, and to improve the success rate of primary cultured. The use of fresh Schwann cell tumor tissue samples collected 10 cases of type Ⅱ neurofibromatosis patients for the experimental material, I collagenase digestion culture extracts of neoplastic Schwann cells, low concentrations of trypsin digestion quickly, the difference The purified cells adherent law and clone screening technology. Endoscopic morphologic, the combined immunocytochemical method to detect the expression of S-100 protein identification purified cell neoplastic Schwann cells cultured in vitro. The results showed: 10 cases of tumor tissue samples were successfully cultured neoplastic Schwann cells by a total of five cases. Neoplastic Schwann cells in primary culture to three days, see a large number of bipolar spindle-shaped or triangular cells; quickly digest the law and the difference between the number of adherent purified cells with low concentrations of trypsin; training to 3rd generation inverted phase contrast microscope. seen 4 to 5 cell clone, Immunocytochemistry S-100 protein positive expression of neoplastic Schwann cell purity gt; 95%, good vitality can be stabilized subculture. Part II: Building neurofibromatosis type Ⅱ clinically applicable methods of molecular genetic analysis, molecular genetic diagnosis of the type Ⅱ neurofibromatosis patients and their descendants, to guide the of type Ⅱ neurofibromatosis family genetic counseling and individual of condition monitoring, follow-up and clinical intervention. Schwann cells and blood of patients with the primary cultured tumor genomic DNA was extracted; 10 cases of patients with neurofibromatosis type Ⅱ, in two cases of offspring and informed consent to gene sequencing, while the blood of their offspring genomic DNA extraction; paternity second generation of blood and patients neoplastic Schwann cell genomic DNA the NF2 gene sequencing (E1-15 and E17 exons and adjacent intron); according to the patient's clinical manifestations and symptoms develop a reasonable clinical intervention and follow-up plan, combined with the paternity second generation molecular genetic test results, given genetic counseling of the type Ⅱ offspring neurofibromatosis suffering from high-risk populations, and the development of future generations of the follow-up plan. This study starts Section Ⅱ neurofibromatosis tumor specimen bank of genomic DNA library; gene sequencing results showed that neoplastic Schwann cells extracted from the patient's blood NF2 gene mutation; first group of mothers and daughters exist identical the gene mutated sites, gene mutation point of the second group mother is not entirely consistent, detected mutation sites are located near the regulatory region of the exon. Conclusion: 1. Applications were cultured in vitro method combined with a low concentration of enzyme digestion quickly, the difference between the number of adherent method and clone screening, can be effectively removed the Schwann cells in the tumor tissue fibroblasts, high purity, vitality, good tumor Schwann cells, can be used for the incidence and treatment of type Ⅱ neurofibromatosis. 2 type II nerve fibers matosis Schwann cell tumor tissue samples and genomic DNA library organization and genomic DNA resources for the disease molecular genetic research; molecular genetic analysis method can be very good guidance disease clinical work, ahead of the expected offspring cancer risk, to develop individualized follow-up plan for high-risk populations, early detection and early treatment of the disease do; establish a long-term follow-up plan with the patient, to give the individual condition monitoring and clinical intervention can improve patients' quality of life and prolong survival.

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CLC: > Medicine, health > Oncology > Nervous system tumors > Intracranial tumors and brain tumors
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