肿瘤坏死因子相关凋亡诱导配体（TRAIL）的临床前抗肿瘤作用研究

发布时间：2018-05-04 12:31

本文选题：rhTRAIL + 工业生产　；参考：《浙江大学》2014年博士论文

【摘要】：1.pHS-TRAIL表达质粒的构建人肿瘤坏死因子相关凋亡诱导配体(human TNF-Related Apoptosis Inducing Ligand, hTRAIL)是一种跨膜蛋白,由281个氨基酸组成,其中1-14位氨基酸为胞内区,15-40位氨基酸为跨膜区,41-281位氨基酸为胞外区。由于114-281位氨基酸的胞外区片段完全具有促细胞凋亡活性,同时完整的rhTRAIL蛋白分子在体外较难获得表达,因此我们选择114-281位氨基酸所在肽段进行重组表达质粒的构建。先前的研究发现,rhTRAIL表达系统的选择具有一定的难度,若使用真核系统对rhTRAIL蛋白进行表达,容易形成没有生物学活性的二聚体形式；当采用常规的原核系统例如T7表达体系和温度诱导性表达体系时,rhTRAIL蛋白又容易形成包涵体,这些表达系统的问题会给后期纯化工艺、产品质量等均会带来问题,因而我们选用了pBR322载体,并以此为基础构建了一个色氨酸启动子驱动目的基因的载体,随后在色氨酸启动子的下游插入编码hTRAIL114-281片段的基因序列,再将该重组质粒导入宿主菌W3110。构建获得表达载体,确认目的蛋白的正确表达,并初步摸索了其在摇瓶水平影响表达的条件。 2. rhTRAIL的发酵及纯化工艺研究上一章节的研究中,通过基因工程技术将人肿瘤坏死因子相关凋亡诱导配体(Recombinant Human TNF-related apoptosis-inducing ligand、rhTRAIL)编码胞外部分114-281aa的基因片段插入到表达载体pHS色氨酸启动子的下游,得到重组质粒pHS-TRAIL,而后将此质粒转化到W3110菌株内,基因测序结果显示插入的目的基因序列正确。由于rhTRAIL的临床使用剂量可能较大(通常百毫克级),因此对于表达系统、高密度发酵工艺以蛋白纯化工艺的选择和优化以及质量控制均提出了较高的要求。通常,影响大肠杆菌高密度发酵的因素主要包括细胞新陈代谢所需的营养的供给和控制、毒副产物(乳酸和氨)的控制或排出、氧气的供给以及关键参数,如pH值、溶氧浓度和温度等的实时控制问题。本章就rhTRAIL的摇瓶培养、发酵生产工艺以及纯化工艺进行了初步的研究和优化,通过对培养基的组成、高密度发酵条件的优化以及纯化工艺的摸索,从而研究如何在保证生物制品产品质量的前提下提高目的蛋白表达量和纯化工艺的收率,并最终提高rhTRAIL的产量。 3. rhTRAIL的体外及体内药效学及机理研究在前面的研究中,我们通过对培养基的组成、高密度发酵条件的优化以及纯化工艺的摸索,在保证rhTRAIL蛋白品质的前提下提高了蛋白产量,获得了足够量的rhTRAIL蛋白纯品。本章节我们进一步对获得的重组蛋白进行体外及体内药效学及机理研究。由于重组TRAIL蛋白具有广谱抗肿瘤活性,因此我们将HCT-116、Colo-205、A549及MCF-7等12种肿瘤细胞作为体外药效活性研究对象,并且构建了人结肠癌HCT-116和Colo-205裸小鼠模型和非小细胞肺癌95D裸小鼠模型,采用单独用药、联合用药的方式进行了体内药效学研究；最后以A549细胞为实验对象,通过Annexin-PI双染和Western blot的实验手段研究了rhTRAIL蛋白的诱导细胞凋亡途径。 4. rhTRAIL在大鼠体内的药动学和组织分布研究在前述章节中,我们已经利用基因工程手段获得了具有生物学活性的rhTRAIL纯品；本章中,我们研究了大鼠静脉注射5、10和30mg/kg rhTRAIL后,血浆浓度经时变化曲线,并研究了大鼠静脉注射10mg/kg rhTRAIL后,各组织中的rhTRAIL分布情况。结果显示在5-30mg/kg范围内,主要药物动力学参数AUC及Cmax与剂量呈线性相关,CL和V1在三个剂量组没有显著性差异(P0.05),雌雄大鼠间各动力学参数没有显著性差异,同时,大鼠静脉注射rhTRAIL后,其迅速分布到各组织。肾脏为最主要的分布器官,其次为脾、血、卵巢、肝脏和肺,心、脑、脂肪、胃、肌肉、肠、胸腺和睾丸组织分布较低。上述组织中rhTRAIL含量随着时间的延长迅速下降,未观察到蓄积现象。结论肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis inducing ligand, TRAIL)属于肿瘤坏死因子家族,在体内以三聚体的形式可与不同受体结合后通过不同信号通路介导细胞凋亡。相关研究证实TRAIL蛋白对多种人肿瘤细胞(如肺癌、肝癌、结肠癌、白血病细胞等)具有生长抑制和细胞毒效应。国内外一些制药企业均对TRAIL开展了多项临床前和临床研究,但进入临床后的研究进展则较为缓慢,对其原因也未见明确报道。本研究在构建了重组TRAIL蛋白的表达体系并通过工艺优化获得了高纯度的重组TRAIL蛋白后,对其体外、体内药效以及体内代谢、组织分布性质开展了相关研究。研究结果显示重组TRAIL蛋白在体内体外对不同肿瘤细胞均具有一定的生长抑制作用,体内代谢组织分布结果显示其在体内主要分布于肾脏。上述结果一方面提示重组TRAIL蛋白具有良好的成药性,另一方面可能由于TRAIL蛋白被肾小球快速滤过所致的半衰期较短,提示联合小分子用药或TRAIL蛋白偶联小分子药物可能更适合于该分子作为治疗性蛋白药物的开发方向。
[Abstract]:Construction of 1.pHS-TRAIL expression plasmid
The human tumor necrosis factor related apoptosis inducing ligand (human TNF-Related Apoptosis Inducing Ligand, hTRAIL) is a transmembrane protein consisting of 281 amino acids, of which 1-14 amino acids are intracellular, 15-40 amino acids are transmembrane regions and 41-281 amino acids are extracellular domain. The extracellular domain of 114-281 amino acids is fully promoted. The cell apoptosis activity, and the complete rhTRAIL protein molecules are difficult to express in vitro, so we choose the 114-281 amino acid peptide segment for the construction of recombinant expression plasmid. Previous studies have found that the selection of rhTRAIL expression system is difficult. If the eukaryotic system is used to express rhTRAIL protein, it is easy to form. There is no biological activity in the form of two polymer; when the conventional prokaryotic system such as the T7 expression system and the temperature induced expression system are used, the rhTRAIL protein is easy to form inclusion bodies. The problems of these expression systems will bring problems to the later purification process and the quality of the products. Therefore, we choose the pBR322 carrier and take this as the basis. The carrier of a tryptophan promoter to drive the target gene was constructed, then the gene sequence of the encoded hTRAIL114-281 fragment was inserted into the downstream of the tryptophan promoter, and the recombinant plasmid was introduced into the host bacterium W3110. to construct the expression vector, to confirm the correct expression of the target protein, and to preliminarily explore the expression of the target protein at the shake flask level. Conditions.
Study on the fermentation and purification process of 2. rhTRAIL
In the last chapter, the recombinant plasmid pHS-TRAIL was inserted into the downstream of the expression vector pHS tryptophan promoter by the gene engineering technique, which was encoded by the human tumor necrosis factor related apoptosis inducing ligand (Recombinant Human TNF-related apoptosis-inducing ligand, rhTRAIL), and then the recombinant plasmid pHS-TRAIL was obtained. The gene sequencing results show that the inserted target gene sequence is correct. Because the clinical dosage of rhTRAIL may be larger (usually 100 milligram), the high density fermentation process has high requirements for the selection and optimization of protein purification process and quality control for the expression system. Usually, the high density fermentation process has a great influence on the expression system. The factors for high-density fermentation of Enterobacteriaceae include the supply and control of nutrients needed for cell metabolism, control or discharge of toxic by-products (lactic acid and ammonia), supply of oxygen and key parameters, such as pH, dissolved oxygen concentration and temperature. This chapter deals with the shake flask culture, fermentation process and purification process of rhTRAIL. The preliminary study and optimization were carried out. Through the composition of the medium, the optimization of the high density fermentation conditions and the exploration of the purification process, the yield of the target protein expression and the purification process was improved on the premise of ensuring the quality of the biological products, and the production of rhTRAIL was finally raised.
Pharmacodynamics and mechanism of 3. rhTRAIL in vitro and in vivo
In the previous study, we improved the production of protein by improving the composition of the medium, the optimization of high density fermentation conditions and the purification process. We obtained a sufficient quantity of rhTRAIL protein in the premise of guaranteeing the quality of rhTRAIL protein. In this chapter, we further studied the pharmacodynamics of the obtained recombinant protein in vitro and in vivo and in vivo. As a result of the broad-spectrum anti-tumor activity of recombinant TRAIL protein, 12 tumor cells, such as HCT-116, Colo-205, A549 and MCF-7, were used as research objects in vitro, and a nude mouse model of HCT-116 and Colo-205 in human colon cancer and a nude mouse model of non-small cell lung cancer were constructed. In vivo pharmacodynamics study was conducted. Finally, the apoptosis pathway induced by rhTRAIL protein was studied by the experimental means of Annexin-PI double staining and Western blot using A549 cells as the experimental object.
Pharmacokinetics and tissue distribution of 4. rhTRAIL in rats
In the foregoing section, we have obtained the biological activity of rhTRAIL pure products using genetic engineering. In this chapter, we studied the time variation curve of plasma concentration after intravenous injection of 5,10 and 30mg/kg rhTRAIL in rats, and studied the distribution of rhTRAIL in each tissue after intravenous injection of 10mg/kg rhTRAIL. The main pharmacokinetic parameters, AUC and Cmax, were linearly related to the dose in the 5-30mg/kg range. There was no significant difference between the three doses of CL and V1 (P0.05). There was no significant difference in the kinetic parameters between the male and female rats. At the same time, after the intravenous injection of rhTRAIL, the rats were rapidly distributed to the tissues. The kidney was the most important distribution organ. The distribution of the spleen, blood, ovary, liver and lung, heart, brain, fat, stomach, muscle, intestines, thymus and testis was low. The content of rhTRAIL in the above tissues declined rapidly with the time, and the accumulation was not observed.
conclusion
The tumor necrosis factor related apoptosis inducing ligand (TNF-related apoptosis inducing ligand, TRAIL) belongs to the tumor necrosis factor family, which can be combined with different receptors in the form of a tripolymer in the body to mediate cell apoptosis through different signaling pathways. The relevant studies have proved that TRAIL egg white has a variety of human tumor cells (such as lung cancer, liver cancer, and colon cancer). A number of pharmaceutical companies both at home and abroad have carried out a number of pre clinical and clinical studies on TRAIL, but the progress of the study after entering the clinic is slow, and the reasons for it have not been clearly reported.
In this study, the recombinant TRAIL protein expression system was constructed and a highly purified recombinant TRAIL protein was obtained by process optimization. In vitro, in vivo, in vivo, in vivo and in vivo metabolism and tissue distribution properties were studied. The results show that recombinant TRAIL protein has a certain growth inhibition on different tumor cells in vivo. The results showed that it was mainly distributed in the kidneys in vivo. The results suggested that the recombinant TRAIL protein had good drug resistance, and on the other hand, the half-life of TRAIL protein was caused by rapid glomerular filtration, suggesting that small molecular drugs or TRAIL protein coupling small molecular drugs can be used. It is more suitable for the development of this molecule as therapeutic protein drug.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R969.1

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