Functional analysis of malignant brain tumor related gene <h-neu> and development of a novel molecular tageting therapy based on the h-neu gene function

我们研究了一个新基因< h-neu>的产物的功能分析。我们也尝试了一些实验来研究该基因产物在恶性胶质瘤细胞中的作用。首先，我们自制了一种新的抗体来识别h-neu蛋白的表位。由于我们可以证实这种抗体在免疫组织化学中起作用，我们试图对每个级别的胶质瘤样本进行染色。但是我们并没有发现在染色灵敏度上有明显的差异，因此我们认为h-neu在胶质瘤中的表达水平并没有那么高。接下来，我们对每个级别的胶质瘤样本进行Real-Time PCR。h-neu mRNA在恶性胶质瘤中的表达低于低级别胶质瘤。为了研究减少h-neu的产物对恶性胶质瘤生长速度的影响，我们制备了h-neu的siRNA，并将其转染到几个胶质瘤培养细胞中。转染h-neu siRNA对胶质瘤细胞株生长速率的影响是不同的。我们认为胶质瘤细胞系中生长降低率的变化基本是由于h-neu在各细胞系中的天然表达所致，因此我们进行了northern blot分析，测量了h-neu在各细胞系中的表达水平。而在胶质瘤细胞株中，h-neu的表达与siRNA对细胞生长的抑制率无相关性。我们认为我们应该尝试研究h-neu蛋白的功能，我们已经开始使用蛋白质组程序对h-neu进行功能分析。我们现在正在制备h-neu过表达的胶质瘤细胞系。此外，我们仍在计划几个实验来研究h-neu函数。我们想知道h-neu的正常功能，然后我们想尝试了解h-neu功能在胶质瘤细胞系中的意义。

We have been investigated about the analysis of the function in the product of a novel gene < h-neu>. We also have tried several experiments to investigate how this gene prduct functions in the malignant glioma cells. First, we made a new antibody to recognize the epitope of the h-neu protein by ourselves. Since we could confirm that this antibody worked in immunhistochemistry, we tried to stain the tumor samples in every grade of gliomas. However, we could not find out the significant differences in the sensitivity of the staining, therefore, we concluded that the expression level of h-neu in glioma was not so high. Next, we performed a Real-Time PCR in every grade of glioma samples. The expression of the mRNA of h-neu in malignant glioma was low compared as those of low grade gliomg. To investigate how the reduction of the product of h-neu affect the growth rate of malignant gliomas, we made a siRNA of h-neu and performed the transfection of it to several glioma culture cells. The reduction of the growth rate due to the transfection of h-neu siRNA was various in glioma cell lines. We considered that the variety of the reduction rate of growth in glioma cell lines was basically due to the native expression of h-neu in each cell lines, therefore, we performed the Nothern blot analysis and measured the expression level of h-neu in each cell lines. However the there was no relation between the expression of h-neu and the reduction rate of the growth due to siRNA in glioma cell lines. We considered that we should attempt to investigate the function of h-neu protein, we have started the functional analysis of h-neu using the proteomix procedure. We are now preparing the glioma cell line in which h-neu was overexpressed. Furthermore, we are still planning several experiments to investigate the h-neu function. We would like to know the normal function of h-neu and after that we would like to try to know the meaning 3 of h-neu function in glioma cell lines.