CN117677692A - Cell strain for TGF beta detection and high-precision TGF beta detection method - Google Patents
Cell strain for TGF beta detection and high-precision TGF beta detection method Download PDFInfo
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Abstract
The invention discloses a cell strain for TGF beta detection and a high-precision TGF beta detection method, in particular to a cell strain for TGF beta detection in human body samples, which is created by screening human cells with TGF beta response of more than 300 times. The method can be used for detecting the human body sample and is not influenced by the human body sample. Compared with ELISA detection method, the sensitivity is improved by 30 times, the TGF beta content of 1pg/ml can be detected, and meanwhile, the detection method has high selectivity and high reliability on detection molecules. Moreover, the high accuracy and reproducibility of the present invention have been demonstrated by mass detection of cell culture media, mouse serum, human serum and plasma.
Description
The invention relates to a cell strain for TGF beta detection and a high-precision TGF beta detection method, which can be used for early auxiliary detection and prevention of autoimmune diseases, aging and cancers, and belongs to the technical field of biology.
Autoimmune disease is a disease that produces a false immune response to self-antigens via the human immune system, resulting in damage to tissues in the body. According to the pathology and symptoms, the characteristics of the composition mainly comprise organ specificity and systematicness, and the characteristics comprise local or systemic inflammation, lymphadenectasis, leucopenia in blood and the like. Common autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and the like.
Aging refers to the intrinsic degradation process of the human body, and is divided into two categories, namely physiology and pathology, wherein the physiological degradation is mainly characterized by hypometabolism of cells, change of tissue structure and gradual loss of physiological functions, and the specific physiological manifestations are cardiovascular system hardening, nerve conduction speed reduction, digestion and respiratory function weakening and the like.
Cancer is abnormal proliferation of cells, invasion of surrounding tissues, and systemic spread caused by abnormal cell functions due to a plurality of gene mutations. Early clinical symptoms are usually tumor growth or localized pain, and sometimes no obvious symptoms. In the advanced stages, cancer cells spread through the blood and lymphatic systems, increasing the difficulty of treatment, accompanied by extremely high mortality.
The existing autoimmune diseases, aging and cancer detection mostly use an enzyme-linked immunosorbent assay (ELISA), and the effect of judging the disease condition is achieved by detecting the molecular index in blood. However, ELISA techniques have the disadvantages of low reproducibility, low sensitivity, low selectivity, high detection cost, and great limitations in early detection of diseases. The cost required for a single ELIZA assay is greater than 10 cents, and the sensitivity is above or below 30pg/ml, and the specificity is also poor.
Meanwhile, the prior art researches show that the content of transforming growth factor beta (TGF beta) in serum is obviously changed along with the disease degree in autoimmune diseases, aging and cancers. Mice with lupus erythematosus have lower levels of active tgfβ in serum than healthy individuals; the serum active TGF beta content of the middle-aged mice with aging signs is higher than that of the young mice; mice receiving TGF-beta inhibitory vaccines grew more slowly than mice not receiving the vaccine. Thus, tgfβ can be used as an effective predictor and diagnostic molecule for early detection of autoimmune diseases, aging and cancer.
The method disclosed in New reagents for improved in vitro and in vivo examination of TGF-bsignaling (Growth Factors, october 2011;29 (5): 211-218) cannot be used for detection of human samples, and when used for human samples, the detection sensitivity is significantly reduced.
Disclosure of Invention
The detection method of TGF beta provided by the invention can be used for detecting human body samples, has higher sensitivity, and can provide a more efficient early detection and prevention method for autoimmune diseases, aging and cancers through technical and application innovation, and the problems existing in the existing ELISA technology can be optimized.
It is an object of the present invention to provide a cell line for TGF-beta (transforming growth factor-beta) detection, which is a human cell line having a TGF-beta response of 300-fold or more, or a human cell line having a detection sensitivity of 1pg/ml, which is established after screening.
In a specific example, the cell line for TGF-beta detection in human samples was deposited at Mary Fabricius cell Bank (CellBank Australia, address: australia) Australia, 3.10.2021, accession number CBA20210033.
Another object of the present invention is to provide a TGF-beta detecting cell line for human samples, which is transfected with a reporter gene-containing pCAGA based on the cell strain of the present invention (n) -a reporter virus of luc.
The third object of the invention is to provide the application of the cell strain or the TGF-beta detection cell line in the detection of TGF-beta in human body samples.
The fourth object of the present invention is to provide a method for detecting TGF-beta in a human sample, which comprises the step of detecting TGF-beta in a human sample by using a PCR primer (n) The reporter virus of luc is transfected into the cell lines of the invention and the tgfβ content of the sample is detected by co-culturing the transfected cells with the sample.
The reporter gene pCAGA of the invention (n) Luc is a TGF-beta related reporter gene that has been reported in the prior art, e.g.Ad-CAGA as used in document Live Cell Imaging of the TGF-beta/Smad 3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System 12 Luc reporter, where luc represents luciferase (luc), may be a luciferin commonly used in the art, such as that having NCBI number 249591.
The invention relates to a gene pCAGA containing a reporter gene (n) Construction of the reporter virus of luc the transfection of the reporter virus into the cell lines according to the invention may be carried out according to methods conventional in the art, i.e.the reporter gene pCAGA (n) Transfection of luc into human cell lines according to the invention can be carried out according to methods conventional in the art, for example by the reporter gene pCAGA (n) Cloning the luc into adenovirus expression vector, transfecting the vector into cell strain after restriction enzyme digestion, extracting the reporter virus by lysing the cells, and preparing the vector containing pCAGA (n) The reporter virus of luc is transfected into the human cell line according to the invention.
In some embodiments of the invention, the transfected cells may be co-cultured with the sample to detect TGF-beta content in the sample, e.g., according to methods conventional in the art, e.g., as disclosed in New reagents for improved in vitro and in vivo examination of TGF-beta signaling (Growth Factors, october 2011;29 (5): 211-218). In one embodiment, the specific scheme is as follows:
(1) Detecting TGF beta at a specified concentration and TGF beta in the sample by transfected cells respectively;
(2) And constructing a standard curve according to the detection result of the TGF beta with the specified concentration, and calculating the content of active TGF beta in the sample according to the curve.
In one embodiment of the invention, the sample of the invention is a human body fluid, which may comprise an intracellular fluid or an extracellular fluid, including, but not limited to, a human serum sample.
In one embodiment of the invention, the cell density of the test cell line is 10 to 100 samples per μl.
The cell strain or the detection cell line or the detection method of TGF beta is applied to the preparation of auxiliary diagnostic reagents for cancers and aging autoimmune diseases.
It should be noted that the specific conditions described herein are merely illustrative of the present invention and are not intended to limit the present invention.
Experiments show that the method can be used for detecting human body samples and is not influenced by the human body samples. Compared with ELISA detection method, the sensitivity is improved by 30 times, the TGF beta content of 1pg/ml can be detected (as shown in the figures 4 and 5 of the invention), and meanwhile, the detection molecule has high selectivity and extremely high reliability. Moreover, the high accuracy and reproducibility of the present invention have been demonstrated by mass detection of cell culture media, mouse serum, human serum and plasma. The method adopts 96/384 well plates (96/384-well plate), and can complete one detection turnover within 24 hours, thereby greatly increasing the detection efficiency from two dimensions of sample size and time. And the single detection cost is lower than $1, and compared with ELISA technology, the detection time and cost are reduced. TGF beta can be used for the auxiliary diagnosis of autoimmune diseases, aging and cancers because of the variation of the expression of TGF beta in autoimmune diseases, aging and cancers.
FIG. 1 is a comparison of active TGF beta levels in serum of healthy and autoimmune disease-bearing mice;
FIG. 2 is a comparison of active TGF-beta levels in serum from young (10 weeks) and adult (40 weeks) mice;
FIG. 3 is a comparison of active TGF-beta content in serum of vaccinated versus unvaccinated TGF-beta inhibitory vaccine mice;
FIG. 4 is a standard curve constructed by different TGF-beta content versus cellular responses detected by the present invention (left) and ELISA (right);
FIG. 5 shows the detection of TGF-beta content in different human serum samples by the invention (C) and ELISA (B).
FIG. 6 is a standard curve constructed from different TGF-beta levels measured in the control and cell responses.
The invention will be further illustrated with reference to specific examples. The embodiments described herein are provided for illustration and understanding of the invention and are not intended to limit the invention.
In the following examples, the transfection contained pCAGA (n) Cell lines of the reporter virus of luc were deposited at the Mary Fabricius cell Bank (CellBank Australia, address: australia) of Australia at 3.10.2021 under accession number CBA20210033.
EXAMPLE 1 construction of TGF-beta detecting cell lines
(1) Reporter gene pCAGA (n) Cloning of the luc into the entry vector pENTR 1A;
(2) Recombining a reporter gene to an adenovirus expression vector pAd/PL-DEST through a specific site attL-attR;
(3) The adenovirus expression vector pAd/PL-DEST was digested with restriction enzyme PacI;
(4) Transfection of linear pAd/PL-DEST into 293A cell lines;
(5) Lysing 293A cells to extract the reporter virus;
(6) Will contain pCAGA (n) The reporter virus of luc was transfected into the human cell line of the invention (accession number CBA 20210033) at a multiplicity of infection of 200 to obtain a test cell line. The cell line, like the cell line deposited in the present invention, is capable of producing different responses to different TGF beta levels, and is embodied in gene transcription and protein production guided by phosphorylated modified Smad 3.
Example 2 detection of TGF beta at different concentrations and construction of a Standard Curve
(1) TGF-beta was added to DMEM medium at various concentrations. The concentrations used in this example were 0, 0.007, 0.02, 0.06, 0.18, 0.56, 1.67, 5ng/ml.
(2) The test cell line constructed in example 1 was cultured in 96-well plates with the medium supplemented with TGF-beta of step (1) at a density of 5X 10 3 Each hole. After cells reach a fusion state and are incubated for 24 hours, cells are lysed by using a cell lysis buffer, the lysate is centrifuged to extract a supernatant, a luciferin reagent is added, and a fluorometer is used to detect the degree of fluorescence released after the reaction of luciferin with luciferase produced by a reporting system in solution.
(4) According to the detection result and the corresponding TGF beta concentration, as shown in fig. 4, the comparison with the detection by the traditional ELISA method shows that the response of the detection cell line disclosed by the invention and the TGF beta is as low as 300 times or more, the detection accuracy of the TGF beta content in serum can reach 1pg/ml, the detection effect is obvious, and the detection effect cannot be achieved by using the ELISA technology for the same sample.
Comparative example
A test cell line was constructed in the same manner as in example 1, except that the human cell line used was human breast cancer cell MDA-MB-231, which was commercially available, and was tested in the same manner as in example 2, and the results of the test cell line constructed from the cells used in this example showed a minimum 200-250 fold response to TGF-beta, and the accuracy of the test for the TGF-beta content in serum was 5-10pg/ml, which was far lower than that of example 2, as shown in FIG. 6.
Example 3 detection of serum samples for autoimmune diseases
(1) For autoimmune diseases, this example uses C57BL/6 SHIP-1 -/- Mice were compared to healthy mice. SHIP-1 -/- Mice lack 5' inositol triphosphate in blood cells, exhibiting conditions similar to human lupus erythematosus, and are characterized by increased bone marrow stem cell numbers, macrophage activation, increased levels of proinflammatory factors in serum, and B cell overactivation. This example was also compared with healthy Human (HC) serum using 60 autoimmune patient sera. The disease of the patient is Systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), dermatomyositis (DM) or systemic lupus erythematosusScleroderma (SS).
(2) The blood of mice or human body is extracted, and the non-coagulated serum fraction is extracted after precipitation at 4 deg..
(3) 10% (v/v) of the serum sample from step (2) was added to the DMEM medium.
(4) The test cell line constructed in example 1 was cultured in 96-well plates at a density of 5X 10 using the medium of step (3) 3 Each hole. After cells reach a fusion state and are incubated for 24 hours, cells are lysed by using a cell lysis buffer, the lysate is centrifuged to extract a supernatant, a luciferin reagent is added, and a fluorometer is used to detect the degree of fluorescence released after the reaction of luciferin with luciferase produced by a reporting system in solution.
(5) Based on the above detection results, the corresponding positions of the fluorescence degrees caused by different serum samples are marked on the standard curve constructed in example 2, and the TGF-beta content in serum is estimated.
(6) Experimental results: the serum TGF-beta content of mice exhibiting autoimmune disease symptoms is significantly reduced compared with healthy mice (figure 1), and the serum TGF-beta content of autoimmune disease patients is significantly reduced compared with healthy people (figure 5).
This example demonstrates that the cells of the invention can detect different concentrations of tgfβ content and can aid in the prediction and diagnosis of autoimmune disease. The detection cell line provided by the invention has the advantages that the detection accuracy of the TGF beta content in serum reaches 1pg/ml, the detection effect is obvious, the sensitivity is higher, and the detection effect cannot be achieved by using ELISA technology for the same sample (as shown in figures 4 and 5).
Example 4 detection of aged serum samples
For aging, this example was compared using 40 weeks versus 10 weeks C57BL/6 mice.
TGF-beta content was measured as described in example 3, steps (2) through (5), and the results showed that the serum TGF-beta content was significantly increased in 40 week mice compared to 10 week mice (FIG. 2). It can be used for the auxiliary prediction and diagnosis of aging.
Example 5 detection of serum samples for cancer
For cancer, this example uses C57BL/6 mice that received subcutaneous B16-OVA tumor cell injection, and vaccinated versus non-vaccinated TGF-beta inhibitory vaccine mice were compared. Mice injected with tumor cells developed a distinct tumor within two weeks. TGF-beta inhibitory vaccines contain sT beta RII-Fc by a mechanism that blocks free TGF beta factor.
TGF-beta content was measured as described in example 3, steps (2) through (5), and the results indicated that the serum TGF-beta content of mice vaccinated with TGF-beta inhibitory vaccines was significantly reduced compared to non-vaccinated mice (see FIG. 3). It can be seen that it can be used for the auxiliary prediction and diagnosis of cancer. From the results, the serum TGF-beta content detection method based on the standard curve has good autoimmune diseases, aging and cancer prediction and diagnosis efficacy.
Claims (10)
- By screening human cells with TGF-beta response up to 300 times or more, a cell strain for TGF-beta detection in human samples is created.
- A cell line for tgfβ detection in a human sample according to claim 1 wherein the accession number CBA20210033.
- A TGF-beta detecting cell line for human body sample, which is transfected with a cell line according to claim 1 or 2, comprising a reporter gene pCAGA (n) -a reporter virus of luc.
- Use of the cell line of claim 1 or 2 or the tgfβ -detecting cell line of claim 3 in tgfβ detection.
- Use of the cell line of claim 1 or 2, the tgfβ -detecting cell line of claim 3 for the preparation of an auxiliary diagnostic reagent for cancer, aging, autoimmune diseases.
- Method for detecting TGF beta in human body sampleCharacterized in that the gene containing the reporter gene pCAGA (n) -transfection of a luc reporter virus into a cell line according to claim 1 or 2, and detection of tgfβ content in the sample by co-cultivation of the transfected cells with a human sample.
- The method according to claim 5, wherein,(1) Detecting TGF beta at a specified concentration and TGF beta in the sample by transfected cells respectively;(2) And constructing a standard curve according to the detection result of the TGF beta with the specified concentration, and calculating the content of active TGF beta in the sample according to the curve.
- The method according to claim 5, wherein the human sample is a human body fluid.
- The method according to claim 5, wherein the transfected cells are co-cultured with the sample at a cell density of 10 to 100 cells/μl sample.
- The method according to claim 5, wherein the method has a detection sensitivity of 1pg/ml.
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