CN114107494A - 用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用 - Google Patents
用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用。该用于软骨肉瘤诊治的生物标记物为谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA，所述的谷氨酰胺酶基因具有如SEQ ID No.1所示的核苷酸序列。本发明研究发现，谷氨酰胺酶在软骨肉瘤细胞中表达上调，靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性可能就是治疗软骨肉瘤的潜在新靶点，因此，将谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA作为软骨肉瘤诊治的生物标记物，可用于筛查能够靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性的药物，为软骨肉瘤的治疗提供新途径。
Description
技术领域
本发明属于生物医药技术领域，具体涉及一种用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用。
背景技术
软骨肉瘤(Chondrosarcoma，CHS)是一种发生于软骨细胞或成软骨结缔组织的原始间充质细胞或软骨基质胚胎残迹的骨恶性肿瘤，肿瘤细胞只形成软骨样组织，其可由原位的良性肿瘤，如内生性软骨瘤或滑膜软骨瘤恶变而来，产生软骨基质是其疾病特征。据估计，软骨肉瘤的总体发病率约为1/200,000，约占骨恶性肿瘤的20％，是继多发性骨髓瘤和骨肉瘤后的第三大肿瘤，发病年龄在30-60岁之间。
目前软骨肉瘤的临床治疗主要以外科手术为主，早期手术是软骨肉瘤公认最有效的治疗方式。但对于患有不可切除或转移性软骨肉瘤的患者，因其对放化疗不敏感，临床上仍欠缺有效的治疗方法。因此，针对CHS治疗的有效药物仍需进一步开发。
发明内容
本发明的发明目的是提供一种用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用。
为实现上述发明目的，本发明的技术方案如下：
用于软骨肉瘤诊治的生物标记物，该生物标记物为谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA，所述的谷氨酰胺酶基因具有如SEQ ID No.1所示的核苷酸序列。
本发明研究发现，谷氨酰胺酶在软骨肉瘤细胞中表达上调，靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性可能就是治疗软骨肉瘤的潜在新靶点，因此，将谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA作为软骨肉瘤诊治的生物标记物，可用于筛查能够靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性的药物，为软骨肉瘤的治疗提供新途径。
进一步地，本发明提供了谷氨酰胺酶抑制剂在制备治疗软骨肉瘤的药物中的应用。
作为优选，所述的谷氨酰胺酶抑制剂包括替格拉司他，替格拉司他的结构式如式(Ⅰ)所示：
替格拉司他(Telaglenastat)是一种特异性的谷氨酰胺酶抑制剂，本发明的体内外试验数据表明，替格拉司他处理能够显著降低软骨肉瘤细胞中的谷氨酰胺酶活性，抑制软骨肉瘤细胞增殖并促进软骨肉瘤细胞凋亡，进而达到治疗软骨肉瘤的作用；Telaglenastat具有毒性低，效果明显的特点，可以为软骨肉瘤疾病的综合治疗提供一种替代治疗药物。
本发明还提供了谷氨酰胺酶抑制剂联合衣霉素在制备治疗软骨肉瘤的药物中的应用。
其中，所述的谷氨酰胺酶抑制剂包括替格拉司他，替格拉司他的结构式如式(Ⅰ)所示：
所述的衣霉素的结构式如式(Ⅱ)所示：
本发明研究发现，衣霉素能够诱导软骨肉瘤细胞产生内质网应激，使包括谷氨酰胺酶在内的大量未折叠蛋白质堆积，最终引起软骨肉瘤细胞发生凋亡。将衣霉素与替格拉司他联用，一方面，衣霉素不仅能够引起内质网应激，而且还降低了功能性谷氨酰胺酶的产出；另一方面，替格拉司他不仅能够降低谷氨酰胺酶的mRNA的表达水平，而且能够进一步降低已折叠产出的功能性谷氨酰胺酶的活性，二者协同增效，共同诱导软骨肉瘤细胞凋亡。
本发明还提供了一种治疗软骨肉瘤的药物，该药物的有效成分包括谷氨酰胺酶抑制剂；
或者，该药物的有效成分包括谷氨酰胺酶抑制剂和衣霉素。
当有效成分中包含谷氨酰胺酶抑制剂和衣霉素时，作为优选，该谷氨酰胺酶抑制剂包括替格拉司他，且替格拉司他和衣霉素的用量比为100：(2-32)；作为进一步优选，替格拉司他和衣霉素的用量比为100：(4-16)；作为最优选，替格拉司他和衣霉素的用量比为25:4。
在上述的治疗软骨肉瘤的药物中，所述的谷氨酰胺酶抑制剂(即替格拉司他)的用量为15mg/kg。
与现有技术相比，本发明的有益效果体现在：
(1)本发明研究发现，谷氨酰胺酶在软骨肉瘤细胞中表达上调，靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性可能就是治疗软骨肉瘤的潜在新靶点，因此，将谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA作为软骨肉瘤诊治的生物标记物，可用于筛查能够靶向抑制软骨肉瘤细胞中的谷氨酰胺酶活性的药物，为软骨肉瘤的治疗提供新途径。
(2)本发明的体内外试验数据表明，替格拉司他处理能够显著降低软骨肉瘤细胞中的谷氨酰胺酶活性，抑制软骨肉瘤细胞增殖并促进软骨肉瘤细胞凋亡，进而达到治疗软骨肉瘤的作用；替格拉司他具有毒性低，效果明显的特点，可以为软骨肉瘤疾病的综合治疗提供一种替代治疗药物。
(3)本发明研究发现，衣霉素能够诱导软骨肉瘤细胞产生内质网应激，使包括谷氨酰胺酶在内的大量未折叠蛋白质堆积，最终引起软骨肉瘤细胞发生凋亡。将衣霉素与替格拉司他联用，一方面，衣霉素不仅能够引起内质网应激，而且还降低了功能性谷氨酰胺酶的产出；另一方面，替格拉司他不仅能够降低谷氨酰胺酶的mRNA的表达水平，而且能够进一步降低已折叠产出的功能性谷氨酰胺酶的活性，二者协同增效，共同诱导软骨肉瘤细胞凋亡。
附图说明
图1为CCK-8法检测不同浓度Telaglenastat对SW1353细胞的毒性作用；
其中，Tela(μM)表示替格拉司他Telaglenastat(微摩尔)，Cell viability(Abs@450nm)表示细胞活力，下同；
图2为Telaglenastat处理后SW1353细胞形态变化；
图3来源于内生软骨瘤、低级别软骨肉瘤、高级别软骨肉瘤的临床标本的谷氨酰胺酶(GLS1)表达情况；
其中，Enchondroma表示内生软骨瘤样本，Low grade chondrosarcoma表示低级别软骨肉瘤样本，High grade chondrosarcoma表示高级别软骨肉瘤样本；
图4为rt-PCR法检测不同浓度Telaglenastat处理对SW1353细胞内谷氨酰胺酶(GLS-1)mRNA转录水平的影响；
其中，Ralative mRNA expression(GLS-1/β-actin)表示谷氨酰胺酶的相对mRNA表达水平(内参为β-actin)；
图5为流式细胞仪检测0μM Telaglenastat处理后SW1353细胞凋亡水平；
其中，FITC-A表示荧光染料异硫氰酸荧光素，PI-A表示荧光染料溴化乙锭；下同；
图6为流式细胞仪检测50μM Telaglenastat处理后SW1353细胞凋亡水平；
图7为流式细胞仪检测100μM Telaglenastat处理后SW1353细胞凋亡水平；
图8为流式细胞仪检测不同浓度Telaglenastat处理后SW1353细胞凋亡水平的定量分析图；
其中，Apoptotic cell(％)表示凋亡细胞(百分比)，下同；
图9为Western Blot法检测不同浓度Telaglenastat处理后SW1353细胞中凋亡相关蛋白Bax、Bcl-2、c-caspase3的表达变化；
图10为Western Blot法检测不同浓度Telaglenastat处理后SW1353细胞中凋亡相关蛋白Bax、Bcl-2、c-caspase3表达水平的定量分析图；
其中，The level of protein expression(of control)表示蛋白的表达水平(与对照组相比)；
图11为持续腹腔注射Telaglenastat后小鼠体内软骨肉瘤的体积变化；
其中，Chondrosarcoma Volume表示软骨肉瘤体积，Telaglenastat表示替格拉司他处理组，Saline表示生理盐水处理组，Time(Days)表示处理时间(天数)，Volume(mm3)表示体积(立方毫米)；
图12为注射Telaglenastat 10天/20天后从小鼠体内取出的软骨肉瘤组织图片；
其中，Telaglenastat表示替格拉司他处理组，PBS表示PBS处理组，10days表示10天，20days表示20天；
图13为不同浓度衣霉素处理对SW1353细胞凋亡水平的影响；
其中，TM(μM)表示衣霉素tunicamycin(微摩尔)，下同；
图14为衣霉素处理对SW1353细胞中内质网应激标记物GRP78蛋白表达水平的影响；
图15为Telaglenastat与衣霉素联合处理对SW1353细胞凋亡水平的影响。
具体实施方式
下面结合附图和具体实施方式对本发明的技术方案做进一步详细说明。
实施例1 Telaglenastat对软骨肉瘤细胞凋亡的诱导作用
本实施例利用Telaglenastat(购自上海芮晖化工科技有限公司)处理软骨肉瘤SW1353细胞系(购自美国典型培养物保藏中心ATCC)，以了解Telaglenastat对软骨肉瘤SW1353细胞凋亡的诱导作用。试验主要从以下几个方面进行：
(1)检测Telaglenastat对SW1353细胞的细胞毒性
为了评估Telaglenastat对SW1353细胞的增殖毒性作用，用逐渐增加的浓度(0，5，10，20，50，100μM)的Telaglenastat处理软骨细胞24小时，并使用细胞计数试剂盒-8(CCK-8；Dojindo)检测细胞增殖活性。
检测方法包括：将SW1353细胞接种至96孔板(5*104个细胞/cm2)中培养24小时，然后用不同浓度Telaglenastat处理SW1353细胞24小时后，用PBS洗涤细胞，然后加入100μl含有10μl的CCK-8溶液的高糖DMEM培养基，每孔在37℃下孵育2小时。用全自动定量绘图酶标仪(Thermo)在450nm处测量孔的吸光度，检测结果见图1。由图1可见，随着Telaglenastat浓度的增加，Telaglenastat对SW1353细胞的增殖毒性作用增强，IC50约为100μM。
接着，通过显微镜观察经相同处理时间后，0μM Telaglenastat处理组与50μMTelaglenastat处理组的细胞形态。检测方法包括：SW1353细胞经传代、以105个细胞/cm3的浓度接种在6孔板中培养24小时，然后用指定浓度的Telaglenastat处理细胞24小时后，使用显微镜(Nikon)在明场视野下进行拍摄，视野随机挑选；拍摄结果如图2所示。
由图2可见，在相同处理时间下，与0μM Telaglenastat处理组相比，50μMTelaglenastat处理组的细胞形态明显变圆，凋亡细胞数量增加。
(2)检测Telaglenastat对谷氨酰胺酶GLS-1转录水平的影响
为验证软骨肉瘤的恶性程度与谷氨酰胺酶的表达的潜在关系，我们从临床患者肿瘤组织中获得相应内生软骨瘤、低级别软骨肉瘤、高级别软骨肉瘤的组织标本并进行组织化学染色。
免疫组织化学染色的方法为：从临床患者中获得相应样本，经4％多聚甲醛固定、脱水、石蜡包埋后制备4μm厚度的标本切片，而后进行脱蜡处理，PBS洗涤脱蜡切片后，用0.3％的氢过氧化物孵育10min以阻断内源性的过氧化物酶活性，而后用5％牛血清白蛋白、37℃孵育10min；接着，将切片与含有相应一抗的PBS稀释液(1：100)在4℃环境下孵育过夜；随后，用PBS洗涤3次，将切片与含有相应二抗的PBS稀释液反应20min，并用PBS洗涤3次，再用苏木素复染细胞核；最后用光学显微镜观察，并拍照记录。继续对谷氨酰胺酶高表达细胞在总细胞中的占比进行检测，检测结果见图3。
如图3所示，随着肿瘤的恶性程度增加，细胞中谷氨酰胺酶的表达量明显提升，提示GLS1可能是软骨肉瘤疾病发生发展的重要因素。
在此基础上，进一步分析Telaglenastat对谷氨酰胺酶GLS-1转录水平的影响。检测方法包括：收集0μM Telaglenastat处理组、50μM Telaglenastat处理组和100μMTelaglenastat处理组的SW1353细胞，用TRIzol法(Invitrogen)提取细胞总RNA；而后用PrimeScript-RT试剂盒(TAKA RA)，使用CFX96实时PCR系统(Bio-Rad)反向转录1000ng的总RNA合成cDNA(Fermantas)；并利用CFX96实时PCR系统(Bio-Rad)，用SYBR预混Ex Taq(TAKARA)进行cDNA的扩增。收集周期阈值(Ct)，内参基因(GAPDH)半定量；用2-△△Ct方法评价不同组基因的表达；检测结果如图4。
由图4可见，随着Telaglenastat使用剂量的增加，GLS-1的mRNA表达量下降。
(3)检测Telaglenastat对SW1353细胞凋亡的诱导作用
检测方法包括：SW1353细胞经传代、以105个细胞/cm3的浓度接种在6孔板中培养24小时；而后分别用0μM、50μM和100μM的Telaglenastat处理细胞24小时后，PBS洗涤3次，之后使用细胞凋亡检测试剂盒(南京建成)检测细胞凋亡水平变化，最后通过流式分析仪检测凋亡比率。检测结果如图5、图6、图7和图8所示。
由图5至图8可见，Telaglenastat的使用可以明显促进SW1353细胞的凋亡，且具有剂量依赖性。
(4)检测Telaglenastat对SW1353细胞中凋亡相关蛋白表达水平的影响检测方法包括：
分别提取0μM Telaglenastat处理组、50μM Telaglenastat处理组和100μMTelaglenastat处理组的SW1353细胞中的促凋亡蛋白Bax、c-caspase3，抗凋亡蛋白Bcl-2，以及内参β-actin；用BCA法测定上述蛋白的浓度，根据测定结果取适量上清加入5×Loading buffer与水，使蛋白量定量为30μg/20μL，且5×Loading buffer稀释至1×，并以100℃加热10min使蛋白变性；在SDS-PAGE凝胶的每个孔内加入20μl变性后的蛋白液，电泳后转膜至PVDF膜，5％脱脂奶粉室温封闭2h，TBST洗涤5min×4次后，孵Bax(1：1000)、Bcl-2(1：1000)、c-caspase3(1：1000)andβ-actin(1:2000)作为一抗，4℃孵育过夜。次日，TBST洗涤PVDF膜5min×4次，辣根过氧化酶标记的二抗(二抗：TBST＝1：1000，Bioworld)室温孵育2h，TBST洗涤5min×4次，ECL系统显影。β-actin作为内参，Image lab3.0采集条带灰度值，取目的条带吸光度与内参条带比值作为相对表达量。检测结果见图9和图10。
由图9和图10可见，Telaglenastat的使用可以显著提高促凋亡蛋白Bax、c-caspase3的表达，同时降低抗凋亡蛋白Bcl-2的蛋白水平。
上述试验结果表明，Telaglenastat能够明显抑制SW1353细胞中GLS-1表达同时促进肿瘤细胞的凋亡。
(5)Telaglenastat的体内抗软骨肉瘤作用
为了研究Telaglenastat在体内抗软骨肉瘤作用是否与体外实验结果一致，我们在裸鼠中进行了成瘤实验。
体内实验造模方法包括：
60只雄性裸鼠(浙江大学实验动物中心提供)，5-8周龄，体重18-22g，实验前在动物房适应1周；培养SW1353细胞系，消化离心后重悬达到4*107/ml浓度；用75％乙醇消毒小鼠腹部皮肤，每只裸鼠皮下接种0.1ml；10天后，开始进行药物干预：①实验组：以15mg/kg/day的剂量腹腔注射Telaglenastat；②对照组：注射等量的生理盐水。20天后处死小鼠，取出皮下成型的瘤状组织，并进行体积测量，测量结果如图11和图12所示。
由图11可见，连续注射Telaglenastat明显抑制了SW1353软骨肉瘤细胞在体内的成瘤过程，抑制了肿瘤的生长。并且，如图12所示，在处理的第10天和第20天取出的体内瘤状组织中，Telaglenastat处理组的瘤状组织体积明显小于对照组。由此表明，Telaglenastat在小鼠体内同样具有显著的抗软骨肉瘤作用。
实施例2衣霉素对软骨肉瘤细胞凋亡的诱导作用
本实施例利用衣霉素(购自北京博奥派克)处理软骨肉瘤SW1353细胞系，以了解衣霉素对软骨肉瘤SW1353细胞凋亡的诱导作用。试验主要从以下两个方面进行：
(1)检测衣霉素对SW1353细胞凋亡的诱导作用
检测方法包括：SW1353细胞经传代、以105个细胞/cm3的浓度接种在6孔板中培养24小时；而后分别用0μM、2μM、4μM、8μM、16μM和32μM的衣霉素处理细胞24小时后，PBS洗涤3次，之后使用细胞凋亡检测试剂盒(南京建成)检测细胞凋亡水平变化，最后通过流式分析仪检测凋亡比率。检测结果如图13所示。
由图13可见，衣霉素的使用也可以明显促进SW1353细胞的凋亡，且具有剂量依赖性。
(2)检测衣霉素对SW1353细胞中内质网应激标记物GRP78表达水平的影响
检测方法包括：SW1353细胞经传代、以105个细胞/cm3的浓度接种在6孔板中培养24小时；而后采用10μM的衣霉素处理细胞，分别提取不同处理时间下SW1353细胞的GRP78蛋白，通过Western blot检测GRP78蛋白的表达水平；检测结果如图14所示。
由图14可见，随着衣霉素处理时间的延长，SW1353细胞中内质网应激标记物GRP78蛋白的表达水平逐渐上升，表明衣霉素引起细SW1353胞凋亡的机制可能是衣霉素诱导SW1353细胞产生内质网应激，使包括谷氨酰胺酶在内的大量未折叠蛋白质堆积，最终引起软骨肉瘤细胞发生凋亡。
实施例3 Telaglenastat联合衣霉素对软骨肉瘤细胞凋亡的诱导作用
为了了解Telaglenastat和衣霉素在诱导软骨肉瘤细胞凋亡方面是否存在协同作用，本实施例采用Telaglenastat和衣霉素同时处理SW1353细胞，以观察不同处理条件下SW1353细胞的凋亡水平。
检测方法包括：SW1353细胞经传代、以105个细胞/cm3的浓度接种在6孔板中培养24小时；而后分别用100μM Telaglenastat+2μM衣霉素、100μM Telaglenastat+4μM衣霉素、100μM Telaglenastat+8μM衣霉素、100μM Telaglenastat+16μM衣霉素、100μMTelaglenastat+32μM衣霉素处理细胞24小时，PBS洗涤3次，之后使用细胞凋亡检测试剂盒(南京建成)检测细胞凋亡水平变化，最后通过流式分析仪检测凋亡比率。检测结果如图15所示。
由图15可见，与单独采用Telaglenastat或衣霉素处理SW1353细胞相比，将Telaglenastat和衣霉素同时处理SW1353细胞后，SW1353细胞凋亡比例增长更加明显，细胞凋亡比例更高。
序列表
<110> 浙江大学
<120> 用于软骨肉瘤诊治的生物标记物及谷氨酰胺酶抑制剂在制备治疗软骨肉瘤药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2010
<212> DNA
<213> 人(human)
<400> 1
atgatgcggc tgcgaggctc ggggatgctg cgggacctgc tcctgcggtc gcccgccggc 60
gtgagcgcga ctctgcggcg ggcacagccc ttggtcaccc tgtgccggcg tccccgaggc 120
gggggacggc cggccgcggg cccggctgcc gccgcgcgac tccacccgtg gtggggcggg 180
ggcggctggc cggcggagcc cctcgcgcgg ggcctgtcca gctctccttc ggagatcttg 240
caggagctgg gcaaggggag cacgcatccg cagcccgggg tgtcgccacc cgctgccccg 300
gcggcgcccg gccccaagga cggccccggg gagacggacg cgtttggcaa cagcgagggc 360
aaagagctgg tggcctcagg tgaaaataaa ataaaacagg gtctgttacc tagcttggaa 420
gatttgctgt tctatacaat tgctgaagga caagagaaaa tacctgttca taaatttatt 480
acagcactca aatctacagg attgcgaacg tctgatccca ggttgaaaga gtgtatggat 540
atgttaagat taactcttca aacaacatca gatggtgtca tgctagacaa agatcttttt 600
aaaaaatgtg ttcagagcaa cattgttttg ttgacacaag catttagaag aaagtttgtg 660
attcctgact ttatgtcttt tacctcacac attgatgagt tatatgaaag tgctaaaaag 720
cagtctggag gaaaggttgc agattatatt cctcaactgg ccaaattcag tcccgatttg 780
tggggtgtgt ctgtttgtac agtagatgga cagaggcatt ctactggaga taccaaagtt 840
cccttctgtc ttcagtcctg tgtaaaacct ttgaaatatg ccattgctgt taatgatctt 900
ggaactgaat atgtgcatcg atatgttgga aaagagccga gtggactaag attcaacaaa 960
ctatttttga atgaagatga taaaccacat aatcctatgg taaatgctgg agcaattgtt 1020
gtgacttcac taataaagca aggagtaaat aatgctgaaa aatttgacta tgtcatgcag 1080
tttttgaata agatggctgg taatgaatat gttggattca gtaatgcaac gtttcagtct 1140
gaaagagaaa gtggagatcg aaattttgca ataggatatt acttaaaaga aaagaagtgt 1200
tttccagaag gcacagacat ggttggtata ttagacttct acttccagct gtgctccatt 1260
gaagtgactt gtgaatcagc cagtgtgatg gctgcgacac tggctaatgg tggtttctgc 1320
ccaattactg gtgaaagagt actgagccct gaagcagttc gaaatacatt gagtttgatg 1380
cattcctgtg gcatgtatga cttctcaggg cagtttgctt tccatgttgg tcttcctgca 1440
aaatctggag ttgctggggg cattctttta gttgtcccca atgttatggg tatgatgtgc 1500
tggtctcctc ctctggataa gatgggcaac agtgttaagg gaattcactt ttgtcacgat 1560
cttgtttctc tgtgtaattt ccataactat gataatttga gacactttgc aaaaaaactt 1620
gatcctcgaa gagaaggtgg tgatcaaagg gtaaagtcag tgataaatct tttgtttgct 1680
gcatatactg gagatgtgtc tgcacttcga agatttgctt tgtcagctat ggacatggaa 1740
cagcgggact atgattctag aacagcactc catgtagctg ctgcagaggg tcatgttgaa 1800
gttgttaaat ttttgctgga agcctgcaaa gtaaaccctt tccccaagga caggtggaat 1860
aacactccca tggatgaagc actgcacttt ggacaccatg atgtatttaa aattctccaa 1920
gaataccaag tccagtacac acctcaagga gattctgaca acgggaagga aaatcaaacc 1980
gtccataaga atcttgatgg attgttgtaa 2010
Claims (10)
1.用于软骨肉瘤诊治的生物标记物，其特征在于，为谷氨酰胺酶、谷氨酰胺酶基因或谷氨酰胺酶mRNA，所述的谷氨酰胺酶基因具有如SEQ ID No.1所示的核苷酸序列。
2.谷氨酰胺酶抑制剂在制备治疗软骨肉瘤的药物中的应用。
3.如权利要求2所述的谷氨酰胺酶抑制剂在制备治疗软骨肉瘤的药物中的应用，其特征在于，所述的谷氨酰胺酶抑制剂包括替格拉司他，替格拉司他的结构式如式(Ⅰ)所示：
4.谷氨酰胺酶抑制联合衣霉素在制备治疗软骨肉瘤的药物中的应用。
5.如权利要求4所述的谷氨酰胺酶抑制联合衣霉素在制备治疗软骨肉瘤的药物中的应用，其特征在于，所述的谷氨酰胺酶抑制剂包括替格拉司他，替格拉司他的结构式如式(Ⅰ)所示：
所述的衣霉素的结构式如式(Ⅱ)所示：
6.一种治疗软骨肉瘤的药物，其特征在于，有效成分包括谷氨酰胺酶抑制剂。
7.一种治疗软骨肉瘤的药物，其特征在于，有效成分包括谷氨酰胺酶抑制剂和衣霉素。
8.如权利要求7所述的治疗软骨肉瘤的药物，其特征在于，所述的谷氨酰胺酶抑制剂包括替格拉司他，替格拉司他和衣霉素的用量比为100：(2-32)。
9.如权利要求8所述的治疗软骨肉瘤的药物，其特征在于，替格拉司他和衣霉素的用量比为100：(4-16)。
10.如权利要求6-9中任意一项所述的治疗软骨肉瘤的药物，其特征在于，所述的谷氨酰胺酶抑制剂的用量为15mg/kg。
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| CN110730664A (zh) * | 2017-03-10 | 2020-01-24 | 卡利泰拉生物科技公司 | 含谷氨酰胺酶抑制剂的组合疗法 |
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| CN110730664A (zh) * | 2017-03-10 | 2020-01-24 | 卡利泰拉生物科技公司 | 含谷氨酰胺酶抑制剂的组合疗法 |
| CN111821302A (zh) * | 2019-04-18 | 2020-10-27 | 正大天晴药业集团股份有限公司 | 用于联合治疗软骨肉瘤的喹啉类化合物 |
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