钙调蛋白磷酸酶（CN）是目前所知唯一受Ca2+/钙调素（CaM）调节的Ser/Thr蛋白磷酸酶，是由催化亚基A（CNA）与调节亚基B（CNB）以1:1的比例组成的异二聚体。CN在体内广泛分布，并参与机体的一些重要的生命调控过程如细胞凋亡、T 细胞活化等。CNB作为CN的调节亚基，分子量小，性质稳定，生物学功能研究发现：CNB具有抑制肿瘤生长的潜能，但其作用机制并不清楚。为了将CNB开发为抗肿瘤药物，本室已构建重组人CNB（rhCNB）的基因工程表达系统，通过对相关工艺的研究，获得了高表达、高纯度的rhCNB，并通过初步的药效药理实验，积累了相关的基本数据。为满足药品中试的要求，并进一步完善rhCNB临床前研发资料，本论文在前人的基础上完成rhCNB的中试制备，对rhCNB的临床前药理学进行研究，并探究其抑瘤的作用机制。1．为满足药品中试的要求，在原来成熟的纯化工艺基础上，我们采用大体积的层析柱以及严格无菌的操作流程，对三批50L发酵罐发酵的菌体分别进行纯化，发现改进放大后的纯化工艺稳定，可制备出高纯度，低内毒素含量，活性稳定的rhCNB产品。2．临床前药理学研究部分：首先进一步确定rhCNB药效，发现其能显著抑制荷S180实体瘤小鼠肿瘤的生长，延长荷H22腹水瘤小鼠的存活期；安全性评价发现：急性毒性实验中，按正常剂量的208倍给药时，小鼠未发生死亡现象。正常小鼠连续14天给药后发现，rhCNB对小鼠的体重、肝指数和脾指数均没有明显的影响，说明rhCNB毒性极低；对免疫功能影响实验发现：rhCNB可一定程度上提高小鼠外周血白细胞数量，显著提高小鼠腹腔巨噬细胞的数量，增强其对绵羊红细胞和中性红的吞噬能力，同时可使小鼠外周血T细胞CD4+/CD8+比值升高，以上均说明rhCNB可增强小鼠的非特异性免疫功能。3．为探究CNB的抗肿瘤机理，本文构建真核表达载体，转染细胞并筛选稳定转染克隆，观察其细胞生物学性状发现：与空载体对照细胞相比，CNB过表达可显著促进HEK293细胞和LO2细胞的增殖。同时CNB稳定转染克隆的非锚定依赖性生长能力增强，对血清的依赖性降低，迁移能力明显增强。即CNB过表达促进了细胞的恶性转化。进一步研究发现：CNB过表达细胞中CNA的表达水平以及CN的活性均没有改变，说明促进细胞恶性转化是CNB的单独功能，与CN无关。通过双向电泳与ESI-TOF/TOF质谱鉴定CNB稳定转染克隆与对照细胞中表达的差异蛋白，发现HSP27与DJ-1两种蛋白在CNB稳定转染克隆中表达上调，而据相关报道这两种蛋白与肿瘤发生关系密切，因此认为HSP27与DJ-1可能参与了CNB诱导的细胞恶化。

Calcineurin (CN) is a Ca2+/calmodulin (CaM)-stimulated protein phosphatase. It is a heterodimeric enzyme consisting of a catalytic A subunit (CNA) and regulatory B subunit (CNB). The enzyme, which is ubiquitously expressed in vivo, is critical for several important intracellular signal-transducing pathways, including apoptosis and T-cell activation. CNB is a small molecule with stable characteristics and can promote the activity of CN，Some research suggested that CNB has the effect of antitumor, but little is known about the mechanism.To develop and apply CNB, our laboratory has constructed the Gene Engineering Expression System of recombinant human CNB (rhCNB), and studied the ferment technique, purify technique, and anticancer potential of rhCNB. This paper did some further research in the medical function of rhCNB, and studied the basis of anticancer effect of it.This paper includes there parts:1. Basis on former research, we improve the purification of rhCNB meeting the pilot scale, by increasing the column-volume and manipulating seriously. We purify rhCNB from cells in 50L fermentator three times respectively. The production is with high purity, low quantity of endotoxins, and stable activity.2. Animal experiment was demonstrated that rhCNB has the inhibition function to S180 solid tumor and H22 liver cancer ascites tumor in mice; Acute toxicity experiment of rhCNB indicated that none of the mice died within 14 days even at 208× normal dose. rhCNB has no impact on index of spleen, index of liver, and body weight of mice, while it can increase the number of PWBCs and peritoneal macrophage cells of mice, and also enhance phagocytic function. The results suggest that rhCNB has low toxicity and can induce nonspecific immunity.3. CNB overexpression promoted proliferation of HEK293 and LO2 cells by comparing with vector control cells. Furthermore, stable CNB transfectants showed dramatically improved growth in soft agar, enhanced migration ability, and reduced serum dependence. But in the progress of transformation, the expression of CNA and the activity of CN kept unchanged. Therefore, CNB, rather than CN, is involved in the proliferation promotion of cells. Subsequently, different expression levels between CNB transfectants and controls were identified using 2-DE and ESI-TOF/TOF. Therein, the expression of heat shock protein 27 (HSP27) and protein DJ-1 increased along with CNB overexpression. They may be involved in CNB-induced transformation.