WO2022241762A1 - 一种TGFβ检测用的细胞株及高精度TGFβ检测方法 - Google Patents

一种TGFβ检测用的细胞株及高精度TGFβ检测方法 Download PDF

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WO2022241762A1
WO2022241762A1 PCT/CN2021/095160 CN2021095160W WO2022241762A1 WO 2022241762 A1 WO2022241762 A1 WO 2022241762A1 CN 2021095160 W CN2021095160 W CN 2021095160W WO 2022241762 A1 WO2022241762 A1 WO 2022241762A1
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tgfβ
detection
cell line
human
sample
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PCT/CN2021/095160
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朱宏剑
盛静逸
伊里亚约瑟芬
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江苏九济华生医药科技研究院有限公司
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Priority to AU2021445820A priority Critical patent/AU2021445820A1/en
Priority to PCT/CN2021/095160 priority patent/WO2022241762A1/zh
Priority to CN202180095412.6A priority patent/CN117677692A/zh
Publication of WO2022241762A1 publication Critical patent/WO2022241762A1/zh

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  • the invention relates to a cell line for TGF ⁇ detection and a high-precision TGF ⁇ detection method, which can be used for early auxiliary detection and prevention of autoimmune diseases, aging and cancer, and belongs to the field of biotechnology.
  • Autoimmune disease is a disease in which the human immune system produces a wrong immune response to self-antigens, resulting in tissue damage in the body. According to its pathology and symptoms, it is mainly divided into two types: organ-specific and systemic. Features include local or systemic inflammation, lymphadenopathy, and leukopenia in the blood. Common autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis.
  • Aging refers to the internal degenerative process of the human body, which is divided into two types: physiological and pathological. It is mainly reflected in the decline of cell metabolism, the change of tissue structure and the gradual loss of physiological functions. The specific physiological manifestations are cardiovascular system sclerosis, nerve conduction velocity Decreased, weakened digestive and respiratory functions.
  • Cancer is the abnormal proliferation of cells, invasion of surrounding tissues, and systemic spread caused by abnormal cell function caused by multiple gene mutations. Early clinical symptoms are usually tumor growth or localized pain, and sometimes there are no obvious symptoms. In advanced stages, cancer cells spread through the blood and lymphatic system, making treatment more difficult and accompanied by a high mortality rate.
  • ELISA enzyme-linked immunosorbent assay
  • TGF ⁇ transforming growth factor ⁇
  • the present invention can optimize the problems existing in the existing ELISA technology, and the TGF ⁇ detection method provided can be used for the detection of human body samples, and has higher sensitivity, and can be used for autoimmune diseases, aging, etc. and cancer to provide more efficient early detection and prevention methods.
  • One of the purposes of the present invention is to provide a cell line for TGF ⁇ (transforming growth factor ⁇ ) detection, which is a human cell line with a TGF ⁇ response of more than 300 times after screening, or detection sensitivity Human cell lines at 1pg/ml.
  • TGF ⁇ transforming growth factor ⁇
  • the cell line used for TGF ⁇ detection in human samples was deposited in Ms. Mary Fairfax Cell Bank (CellBank Australia, address: Australia) on March 10, 2021, Deposit number CBA20210033.
  • the second object of the present invention is to provide a cell line for TGF ⁇ detection of human samples, which is transfected with a reporter virus containing reporter gene pCAGA (n) -luc on the basis of the cell line of the present invention.
  • the third object of the present invention is to provide the application of the cell line or the TGF ⁇ detection cell line of the present invention in the detection of TGF ⁇ in human samples.
  • the fourth object of the present invention is to provide a detection method for TGF ⁇ in human samples, transfect the reporter virus containing the reporter gene pCAGA (n) -luc into the cell line of the present invention, and pass the transfected cells and samples The TGF ⁇ content in the samples was detected by co-cultivation.
  • the reporter gene pCAGA (n) -luc described in the present invention is a reporter gene related to TGF ⁇ that has been reported in the prior art, for example the document Live Cell Imaging of the TGF- ⁇ /Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter
  • the Ad-CAGA 12 -luc reporter used in the System wherein luc represents luciferase (luc), can be luciferase commonly used in the art, for example, luciferin with NCBI number 249591.
  • the construction of the reporter virus containing the reporter gene pCAGA (n) -luc of the present invention can be carried out according to the conventional methods in the art, and the reporter virus can be transfected into the cell line according to the present invention, and can also be transfected according to the conventional adenovirus in the art.
  • the transfection of the reporter gene pCAGA (n) -luc into the human cell line of the present invention can be carried out according to the conventional methods in the art, for example, the reporter gene pCAGA (n) -luc is cloned on the adenovirus expression vector , transfected into the cell line after restriction enzyme digestion, the reporter virus was extracted by lysing the cells, and the reporter virus containing pCAGA (n) -luc was transfected into the human cell line of the present invention.
  • the transfected cells are co-cultured with the sample to detect the TGF ⁇ content in the sample, for example, according to conventional methods in the art, for example, New reagents for improved in vitro and in vivo examination of TGF- ⁇ signalling can be used (Growth Factors, October 2011;29(5):211–218).
  • the specific scheme is as follows:
  • TGF ⁇ at the specified concentration and TGF ⁇ in the sample are detected respectively through transfected cells;
  • the sample described in the present invention is a human body fluid, which may include intracellular fluid or extracellular fluid, for example including but not limited to human serum samples.
  • the cell density of the detection cell line is 10-100 cells/ ⁇ l sample.
  • the method of the present invention can be used for detection of human body samples without being affected by human body samples.
  • the sensitivity is improved by 30 times, and the TGF ⁇ content of 1 pg/ml can be detected (as shown in Figures 4 and 5 of the present invention).
  • it has high selectivity for detection molecules and high reliability.
  • the high precision and reproducibility of the present invention have been confirmed through a large number of tests on cell culture medium, mouse serum, human serum and plasma.
  • This method uses a 96/384-well plate (96/384-well plate), and can complete a detection turnaround within 24 hours, greatly increasing the detection efficiency from the two dimensions of sample size and time.
  • TGF ⁇ changes in autoimmune diseases, aging and cancer, it can be used for auxiliary diagnosis of autoimmune diseases, aging and cancer.
  • Figure 1 is a comparison of active TGF ⁇ levels in the serum of healthy and autoimmune disease mice
  • Fig. 2 is young (10 weeks) and adult (40 weeks) mouse serum active TGF ⁇ content comparison;
  • Figure 3 is a comparison of active TGF ⁇ content in serum of mice vaccinated and not vaccinated with TGF ⁇ inhibitory vaccine
  • Fig. 4 is the standard curve constructed by the different TGF ⁇ content and cell response detected by the present invention (left) and ELISA (right);
  • Fig. 5 is the detection of TGF ⁇ content in different human serum samples by the present invention (C) and ELISA (B).
  • Fig. 6 is a standard curve constructed from different TGF ⁇ contents and cell responses detected in the control example.
  • the cell line transfected with the reporter virus containing pCAGA (n) -luc was deposited in Ms. Mary Fairfax Cell Bank (CellBank Australia, address: Australia) on March 10, 2021, Deposit number CBA20210033.
  • test cell line constructed in Example 1 was cultured in a 96-well plate with the medium added with TGF ⁇ in step (1), with a density of 5 ⁇ 10 3 per well. After the cells reached confluence and were incubated for 24 hours, the cells were lysed with cell lysis buffer, the lysate was centrifuged, the supernatant was extracted, the luciferin reagent was added, and the luciferin and the luciferase produced by the reporter system in the solution were detected by a fluorometer The degree of fluorescence released after the reaction.
  • the human cell line used is human breast cancer cell MDA-MB-231, which can be obtained from the market, and tested according to the same method as in Example 2, and the results are shown in Figure 6
  • the detection cell line constructed by the cells used in this example has a minimum TGF ⁇ response of 200-250 times, and the detection accuracy of TGF ⁇ content in serum is 5-10pg/ml, which is far lower than the results in Example 2.
  • this example uses C57BL/6 class SHIP-1 -/- mice to compare with healthy mice.
  • SHIP-1 -/- mice lack 5'-inositol triphosphate in blood cells and exhibit symptoms similar to human lupus erythematosus, which are manifested in increased number of bone marrow stem cells, activation of macrophages, and increased levels of pro-inflammatory factors in serum and overactivation of B cells.
  • This example additionally uses 60 autoimmune patient sera for comparison with healthy human (HC) sera. The patient's condition was systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), dermatomyositis (DM), or systemic sclerosis (SS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • DM dermatomyositis
  • SS systemic sclerosis
  • step (3) Add 10% (v/v) serum sample of step (2) into DMEM medium.
  • the cells of the present invention can detect different concentrations of TGF ⁇ , which can assist in the prediction and diagnosis of autoimmune diseases.
  • the detection cell line of the present invention has a detection accuracy of 1 pg/ml for TGF ⁇ content in serum, and the detection effect is obvious and the sensitivity is higher, while the same sample cannot achieve the detection effect using ELISA technology (as shown in Figure 4 and Figure 5) .
  • this example uses 40-week versus 10-week-old C57BL/6 mice for comparison.
  • step (2) to step (5) of Example 3 the TGF ⁇ content was detected, and the results showed that the TGF ⁇ content in the serum of the 40-week mice was significantly higher than that of the 10-week mice (as shown in FIG. 2 ). It can be seen that it can be used for auxiliary prediction and diagnosis of aging.
  • this example uses C57BL/6 mice that received subcutaneous injections of B16-OVA tumor cells to compare mice vaccinated with and without a TGF ⁇ inhibitory vaccine. Mice injected with tumor cells developed overt tumors within two weeks. TGF ⁇ inhibitory vaccine contains sT ⁇ RII-Fc, the mechanism of which is to block free TGF ⁇ factor.
  • the TGF ⁇ content is detected, and the results show that the TGF ⁇ content in the serum of the mice inoculated with the TGF ⁇ inhibitory vaccine is significantly lower than that of the unvaccinated mice (as shown in Figure 3) . It can be seen that it can be used for auxiliary prediction and diagnosis of cancer. It can be seen from the above results that the detection method of TGF ⁇ content in serum based on the standard curve has good predictive and diagnostic performance for autoimmune diseases, aging and cancer.

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Abstract

提供一种TGFβ检测用的细胞株及高精度TGFβ检测方法，具体为通过筛选TGFβ应答反应达到300倍以上的人源细胞，创建了一种用于人体样品中TGFβ检测的细胞株。所述的方法可以用于人体样品的检测，不受人体样本的影响。相较于ELISA检测方法提高了30倍的敏感度，可检测1pg/ml的TGFβ含量，同时对检测分子具有高选择性，可靠性极高。并且，通过对细胞培养基、小鼠血清、人体血清及血浆的大量检测，已证实了上述方法的高精准度与复现性。

Description

一种TGFβ检测用的细胞株及高精度TGFβ检测方法 技术领域
本发明涉及一种TGFβ检测用的细胞株及高精度TGFβ检测方法，可用于自身免疫疾病、衰老及癌症的早期辅助检测及预防，属于生物技术领域。
背景技术
自身免疫疾病是经人体免疫系统对自身抗原产生错误免疫反应，导致体内组织损害的一种疾病。根据其病理及症状，主要分为器官特异性与系统性两类，特征包括局部或全身炎症、淋巴结肿大、血液中白细胞减少等。常见的自身免疫疾病有系统性红斑狼疮、类风湿关节炎、多发性硬化症等。
衰老指人体内在性的退化过程，分为生理性与病理性两类，主要体现于细胞新陈代谢能力减退、组织结构的变化与生理功能的逐步丧失，具体生理表现为心血管系统硬化、神经传导速度降低、消化与呼吸功能减弱等。
癌症是由多个基因突变引起的细胞功能异常所导致的细胞不正常增生、周围组织入侵及全身性扩散。早期临床症状通常为肿瘤生长或局部性疼痛，也有时无明显症状。在晚期阶段，癌症细胞通过血液与淋巴系统扩散，治疗难度增加，同时伴随着极高的致死率。
现有的自身免疫疾病、衰老及癌症检测大多使用酶联免疫吸附法(ELISA)，通过检测血液内的分子指标达到判断病情的效果。但ELISA技术的缺陷在于其复现性低、敏感度低、选择性低、检测成本高，在疾病早期检测方面存在较大的局限性。单次ELIZA检测所需成本大于10美金，并且敏感度位于30pg/ml上下，专一性也较差。
同时，现有技术研究表明，在自身免疫疾病、衰老及癌症中，血清内转化生长因子β(TGFβ)的含量随病情程度发生显著变化。相较于健康个体，患红斑狼疮小鼠与人类患者血清内活跃TGFβ含量更低；具有衰老体征的中年小鼠血清内活跃TGFβ含量较年轻小鼠更高；接受TGFβ抑制性疫苗的小鼠肿瘤生长速度相较于未接受疫苗小鼠更为缓慢。因此，TGFβ可作为自身免疫疾病、衰老及癌症早期检测的有效预测及诊断分子。
New reagents for improved in vitro and in vivo examination of TGF-bsignalling(Growth Factors,October 2011；29(5):211–218)中公开的方法，不能用于人体样本的检测，当用于人体样品时，检测灵敏度显著降低。
发明内容
本发明通过技术与应用创新，能够优化现有的ELISA技术所存在的问题，所提供的TGFβ的检测方法，可以用于人体样本的检测，且具有更高的灵敏度，可以为自身免疫疾病、衰老及癌症提供更高效的早期检测及预防方法。
本发明目的之一在于提供一种用于TGFβ(转化生长因子β)检测的细胞株，所述细胞株为经筛选后创建的TGFβ应答反应达到300倍以上的人源细胞株，或者说检测灵敏度在1pg/ml的人源细胞株。
在一种具体的实施例中，所述的用于人体样品中TGFβ检测的细胞株于2021年3月10日保藏于澳大利亚玛丽费尔法克斯女士细胞银行(CellBank Australia，地址：澳大利亚)，保藏编号CBA20210033。
本发明目的之二在于提供一种用于人体样品TGFβ检测细胞系，该细胞系在本发明所述的细胞株的基础上被转染了含报告基因pCAGA (n)-luc的报告病毒。
本发明目的之三在于提供本发明所述细胞株或所述的TGFβ检测细胞系在用于人体样品中TGFβ检测中的应用。
本发明目的之四在于提供一种人体样品中TGFβ的检测方法，将含报告基因pCAGA (n)-luc的报告病毒转染到本发明所述的细胞株中，通过转染后的细胞与样品共培养检测样品中的TGFβ含量。
本发明所述的报告基因pCAGA (n)-luc为现有技术已经报道的与TGFβ有关的报告基因，例如文献Live Cell Imaging of the TGF-β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System中所用的Ad-CAGA 12-luc reporter，其中luc表示荧光素酶(luc)，可以为本领域常用的荧光酶素，例如NCBI编号为249591的荧光酶素。
本发明所述的含报告基因pCAGA (n)-luc的报告病毒的构建可以按照本领域的常规方法，报告病毒转染到本发明所述的细胞株中也可以按照本领域常规的腺病毒转染方法，即将报告基因pCAGA (n)-luc转染到本发明所述的人体细胞株中均可以按照本领域的常规方法，例如将报告基因pCAGA (n)-luc克隆到腺病毒表达载体上，经限制酶酶切后转染到细胞株中，通过溶解细胞提取报告病毒，并将含有pCAGA (n)-luc的报告病毒转染到本发明所述的人体细胞株中。
在本发明的一些实施例中，转染后的细胞与样品共培养检测样品中的TGFβ含量例如可以按照本领域的常规方法，例如可以采用New reagents for improved in vitro and  in vivo examination of TGF-βsignalling(Growth Factors,October 2011；29(5):211–218)中公开的方法。在一种实施例中，具体方案如下：
(1)通过转染后的细胞分别检测指定浓度的TGFβ和样品中的TGFβ；
(2)根据指定浓度的TGFβ的检测结果构建标准曲线，并以该曲线推算样本中活跃TGFβ含量。
在本发明的一种实施例中，本发明所述的样品为人体体液，人体体液可以包含细胞内液或细胞外液，例如包括但不限于人的血清样品。
在本发明的一种实施例中，检测细胞系的细胞密度为10～100个/μl样品。
本发明所述的细胞株或检测细胞系或TGFβ的检测方法在用于制备癌症、衰老自身免疫疾病的辅助诊断试剂中的应用。
需要注意的是，此处描述的具体条件仅用于本发明的方案说明，并不用于限制本发明。
通过实验表明，本发明所述的方法可以用于人体样品的检测，不受人体样本的影响。相较于ELISA检测方法提高了30倍的敏感度，可检测1pg/ml的TGFβ含量(如本发明图4、5)，同时对检测分子具有高选择性，可靠性极高。并且，通过对细胞培养基、小鼠血清、人体血清及血浆的大量检测，已证实了本发明的高精准度与复现性。此方法采用96/384孔板(96/384-well plate)，并可在24小时内完成一次检测周转，从样本量及时间两个维度大幅增加了检测效率。且单次检测费用低于1美元，对比ELISA技术降低了检测时间与成本。由于TGFβ在自身免疫疾病、衰老及癌症中表达会出现变化，因此可用于自身免疫疾病、衰老及癌症的辅助诊断。
附图说明
图1为健康与患自身免疫疾病小鼠血清内活跃TGFβ含量对比；
图2为年轻(10周)与成年(40周)小鼠血清内活跃TGFβ含量对比；
图3为接种与未接种TGFβ抑制性疫苗小鼠血清内活跃TGFβ含量对比；
图4为通过本发明(左)与ELISA(右)检测的不同TGFβ含量与细胞反应构建的标准曲线；
图5为通过本发明(C)与ELISA(B)检测不同人体血清样本中的TGFβ含量。
图6对照例检测的不同TGFβ含量与细胞反应构建的标准曲线。
具体实施方式
下面结合具体实施例，对本发明作进一步说明。此处描述的实施例仅用于本发明的说明及理解，并不用于限制本发明。
以下实施例中，被转染含有pCAGA (n)-luc的报告病毒的细胞株，于2021年3月10日保藏于澳大利亚玛丽费尔法克斯女士细胞银行(CellBank Australia，地址：澳大利亚)，保藏编号CBA20210033。
实施例1TGFβ检测细胞系的构建
(1)将报告基因pCAGA (n)-luc克隆到入门载体pENTR1A上；
(2)通过特异位点attL-attR将报告基因重组到腺病毒表达载体pAd/PL-DEST上；
(3)使用限制酶PacI酶切腺病毒表达载体pAd/PL-DEST；
(4)将线性pAd/PL-DEST转染到293A细胞株中；
(5)溶解293A细胞，以提取报告病毒；
(6)将含有pCAGA (n)-luc的报告病毒转染到本发明所述的人体细胞株中(保藏编号CBA20210033)，感染复数为200，获得检测细胞系。此细胞系与本发明保藏的细胞株一样，能够对不同TGFβ含量产生不同的反应，具体体现于由磷酸化修饰的Smad3引导的基因转录与蛋白制造。
实施例2对不同浓度的TGFβ的检测及标准曲线的构建
(1)在DMEM培养基内加入不同浓度的TGFβ。此例中所用浓度为0、0.007、0.02、0.06、0.18、0.56、1.67、5ng/ml。
(2)用步骤(1)加入TGFβ的培养基在96孔板中培养实施例1构建的检测细胞系，密度为5×10 3每孔。细胞达到融合状态并培育24小时后使用细胞裂解缓冲液溶解细胞，将裂解物离心分离后提取上清液，加入荧光素试剂，并使用荧光仪检测荧光素与溶液中报告系统产生的荧光素酶反应后所释放的荧光程度。
(4)根据上述检测结果与对应的TGFβ浓度构建标准曲线，如图4，通过与传统ELISA方法检测的比较发现，本发明所述的检测细胞系与TGFβ应答反应最低达到300倍以上，对血清内TGFβ含量检测精准度可达到1pg/ml，检测效果明显，而相同的样本使用ELISA技术则无法达到检测效果。
对比例
采用实施例1同样的方法构建检测细胞系，区别在于所用的人体细胞株为人乳腺癌细胞MDA-MB-231，可通过市售获得，按照实施例2同样的方法检测，结果如图6 所示，本实施例所用的细胞构建的检测细胞系与TGFβ应答反应最低为200-250倍，对血清内TGFβ含量检测精准度为5-10pg/ml，远低于实施例2的结果。
实施例3针对自身免疫疾病的血清样本进行检测
(1)针对自身免疫疾病，此例先使用C57BL/6类SHIP-1 -/-小鼠与健康小鼠进行比较。SHIP-1 -/-小鼠缺少血液细胞中的5’肌醇三磷酸，表现出类似人类红斑狼疮的病症，具体体现在骨髓干细胞数量增加、巨噬细胞激活、血清内促炎性因子含量增加以及B细胞过度激活。此例另外使用60名自身免疫患者血清与健康人(HC)血清进行比较。患者病症为系统性红斑狼疮(SLE)、类风湿性关节炎(RA)、皮肌炎(DM)或全身性硬化症(SS)。
(2)提取小鼠体内或人体血液，在4°下沉淀后提取未凝固的血清部分。
(3)在DMEM培养基内加入10％(v/v)的步骤(2)的血清样本。
(4)在96孔板中使用步骤(3)培养基培养实施例1构建的检测细胞系，密度为5×10 3每孔。细胞达到融合状态并培育24小时后使用细胞裂解缓冲液溶解细胞，将裂解物离心分离后提取上清液，加入荧光素试剂，并使用荧光仪检测荧光素与溶液中报告系统产生的荧光素酶反应后所释放的荧光程度。
(5)根据上述检测结果，在实施例2所构建的标准曲线上标出不同血清样本导致的荧光程度的对应位置，并推算血清内TGFβ含量。
(6)实验结果：表现自身免疫疾病症状小鼠血清内TGFβ含量相较于健康小鼠有明显下降(如图1)，自身免疫疾病患者血清内TGFβ含量相较于健康人有明显下降(如图5)。
此实施例展现了本发明所述细胞可以检测不同浓度TGFβ含量，可对自身免疫疾病进行辅助预测与诊断。且本发明所述的检测细胞系对血清内TGFβ含量检测精准度达到1pg/ml，检测效果明显，灵敏度更高，而相同的样本使用ELISA技术则无法达到检测效果(如图4、图5)。
实施例4针对衰老的血清样本进行检测
针对衰老，此例使用40周与10周C57BL/6小鼠进行比较。
按照实施例3步骤(2)到步骤(5)的方法进行TGFβ含量检测，结果表明40周小鼠血清内TGFβ含量相较于10周小鼠有明显上升(如图2)。可见，其可用于衰老的辅助预测与诊断。
实施例5针对癌症的血清样本进行检测
针对癌症，此例使用接受皮下B16-OVA肿瘤细胞注射的C57BL/6小鼠，对接种与未接种TGFβ抑制性疫苗的小鼠进行比较。经肿瘤细胞注射的小鼠在两周内发育出明显肿瘤。TGFβ抑制性疫苗内含sTβRII-Fc，其机理为阻塞游离TGFβ因子。
按照实施例3步骤(2)到步骤(5)的方法进行TGFβ含量检测，结果表明接种TGFβ抑制性疫苗的小鼠血清内TGFβ含量相较于未接种疫苗小鼠有明显下降(如图3)。可见，其可用于癌症的辅助预测与诊断。由以上结果可见，以标准曲线作为根据的血清内TGFβ含量检测方法具有很好的自身免疫疾病、衰老以及癌症预测与诊断效能。

Claims (10)

  1. 通过筛选TGFβ应答反应达到300倍以上的人源细胞，创建了一种用于人体样品中TGFβ检测的细胞株。
  2. 根据权利要求1所述的用于人体样品中TGFβ检测的细胞株，其特征在于，保藏编号CBA20210033。
  3. 一种用于人体样品TGFβ检测细胞系，该细胞系在权利要求1或2所述的细胞株的基础上被转染了含报告基因pCAGA (n)-luc的报告病毒。
  4. 权利要求1或2所述的细胞株或权利要求3所述的TGFβ检测细胞系在TGFβ检测中的应用。
  5. 权利要求1或2所述的细胞株、权利要求3所述的TGFβ检测细胞系用于制备癌症、衰老、自身免疫疾病的辅助诊断试剂中的应用。
  6. 一种人体样品中TGFβ的检测方法，其特征在于，将含报告基因pCAGA (n)-luc的报告病毒转染到权利要求1或2所述的细胞株中，通过转染后的细胞与人体样品共培养检测样品中的TGFβ含量。
  7. 根据权利要求5所述的检测方法，其特征在于，
    (1)通过转染后的细胞分别检测指定浓度的TGFβ和样品中的TGFβ；
    (2)根据指定浓度的TGFβ的检测结果构建标准曲线，并以该曲线推算样本中活跃TGFβ含量。
  8. 根据权利要求5所述的检测方法，其特征在于，所述的人体样品为人体体液。
  9. 根据权利要求5所述的检测方法，其特征在于，转染后的细胞与样品共培养时的细胞密度为10～100个/μl样品。
  10. 根据权利要求5所述的检测方法，其特征在于，所述方法检测灵敏度在1pg/ml。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050130193A1 (en) * 2003-09-10 2005-06-16 Luxon Bruce A. Methods for detecting, diagnosing and treating human renal cell carcinoma
WO2006019954A2 (en) * 2004-07-16 2006-02-23 Biogen Idec Ma Inc. In vivo assays for modulation of smad-mediated signaling
CN1906490A (zh) * 2003-11-18 2007-01-31 帝国大学改革有限公司 生物材料及其用途
CN112367994A (zh) * 2018-06-27 2021-02-12 株式会社麦迪帕克特 诊断和治疗表达高水平TGF-β应答标记的癌症患者的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050130193A1 (en) * 2003-09-10 2005-06-16 Luxon Bruce A. Methods for detecting, diagnosing and treating human renal cell carcinoma
CN1906490A (zh) * 2003-11-18 2007-01-31 帝国大学改革有限公司 生物材料及其用途
WO2006019954A2 (en) * 2004-07-16 2006-02-23 Biogen Idec Ma Inc. In vivo assays for modulation of smad-mediated signaling
CN112367994A (zh) * 2018-06-27 2021-02-12 株式会社麦迪帕克特 诊断和治疗表达高水平TGF-β应答标记的癌症患者的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN HAO, WARE THOMAS M.B., IARIA JOSEPHINE, ZHU HONG-JIAN: "Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway <em>In Vitro</em> and <em>In Vivo</em> Using an Adenovirus Reporter System", JOURNAL OF VISUALIZED EXPERIMENTS, no. 137, XP093005682, DOI: 10.3791/57926 *
LUO WEN, HE LI: "Research Progress of Tgf-βand Its Related Dermatosis", CHINESE JOURNAL OF AESTHETIC MEDICINE, vol. 18, no. 9, 30 September 2009 (2009-09-30), pages 1551 - 1552, XP093005686, DOI: 10.15909/j.cnki.cn61-1347/r.2009.10.064 *
LUWOR RODNEY B., WANG BO, NHEU THAO V., IARIA JOSEPHINE, TSANTIKOS EVELYN, HIBBS MARGARET L., SIEBER OLIVER M., ZHU HONG-JIAN: "New reagents for improved in vitro and in vivo examination of TGF-β signalling", GROWTH FACTORS., HARWOOD ACADEMIC PUBLISHERS GMBH., XX, vol. 29, no. 5, 1 October 2011 (2011-10-01), XX , pages 211 - 218, XP009541127, ISSN: 0897-7194, DOI: 10.3109/08977194.2011.615311 *

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