| 摘要: | 生長激素（Growth hormone, GH）是促進人體生長最重要的激素，尤其是對青春期前的兒童更為重要。在臨床治療發現，生長激素缺乏症（Growth hormone deficiency, GHD）的病人在接受生長激素治療後的第一年效果顯著，但生長高度卻因人而異，故認為與個人的遺傳基因特異性有關。我們以單核.酸多型性（Single nucleotide polymorphism, SNP）的方法篩選出適合而且與生長調節有關的遺傳標記。分析的結果顯示GHR codon440（G/T）（氨基酸 Cys/Phe）在統計上有顯著的差異（p＜0.05）。此多型性是核.酸從guanine（G）變成thymine（T），導致在GHR基因上的第440個氨基酸從cysteine變成了phenylananine。為了瞭解這單核.酸多型性所產生的機轉，我們將GHR codon440 G和GHR codon440 T 對於轉譯活性的影響發現T genotype會引起較強的轉譯活性。另一個GHR（GHR-d3多型性）的多型性亦有這樣的結果。然而對於為何這些多型性會造成轉譯活性的增加都尚屬未知。為了瞭解這些問題我們將完成以下五個主要目的：目的一，利用西方墨點法找尋影響轉譯活性的轉譯子。目的二，利用co-immunoprecipitation和bioluminescence resonance energy transfer (BRET)技術來瞭解JAK2, SOCS2和STAT5與GHR間的交互作用；目的三，利用大量表現和RNAi技術抑制SOCS2來瞭解SOCS蛋白對於GHR所引起的轉譯活性的調控；目的四，利用磷酸化蛋白質體學瞭解各GHR間訊號傳遞間的異同；目的五，瞭解GHR cytoplasmic domain的disulfide bond的狀態。藉由這個研究，我們將會更瞭解訊號傳遞的狀態，且進一步瞭解GHR對於細胞的調控機制，將有助於發展更有效的GH對於生長激素缺乏症的治療方式。
Growth hormone replacement is an effective therapy for growth hormone deficient children. However, growth velocity post-growth hormone replacement therapy varies between individuals. In our previous study, we collected a total of 100 growth hormone deficient children (73 boys and 27 girls) who underwent growth hormone therapy for one year to access the possible influences of gene polymorphisms on the growth response to growth hormone in growth hormone deficient children. PCR-RFLP (restriction fragment length polymorphism) and PCR experiments were carried out to detect single nucleotide polymorphisms (SNPs) in the following genes: GHR, JAK2, STAT-5a, STAT-5b, SOCS-2, IGF-1, IGFBP-3, and ALS. We then evaluated the correlations among these gene polymorphisms with various parameters (gender, age, bone age, parents』 heights, serum GH concentration, birth weight) on first year growth velocity. GHR codon 440 G/T (amino acid: Cys to Phe) was the only polymorphism of the GHR gene which correlated with increased growth velocity. The G homozygote was associated with low growth velocity, the G/T heterozygote corresponded with moderate growth velocity and the T homozygote correlated with high growth velocity. The GHR codon 440 T allele showed higher transcriptional activity. Another GHR polymorphism (GHR-d3), exon 3 retention or deletion, was also previously shown to have higher transcriptional activity and associate with first year growth velocity of GHD patients after GH replacement therapy. However, the signaling pathways leads by GHR codon 440 T as well as GHR-d3 to induce the higher transcriptional activity remain largely unknown. In the present project, we proposed to do a comprehensive study on how and why GHR codon 440 T and GHR-d3 induced higher transcriptional activity. This goal will be achieved by completing the five specific aims which are 1) identify the signaling molecule which are responsible for higher transcriptional activity by Western blotting; 2) identify the interaction between SOCS2, JAK2 and STAT5 with GHR by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) technology; 3) identify the SOCS2 regulatory pathway in the GHR induced transcription factor activation by over-expression and RNAi knock-down; 4) using phosphoproteomics approach to identify and characterize the difference in phosphorylated proteins between the wild type and GHR codon 440 T or GHR-d3 ; 5) determine the disulfide bonds in the cytoplasmic domain of GHR. Improving knowledge of how GH act on cells to develop growth promotion activities that lead to help the development of better GH treatments for GHD patients. |
