AU2021445820A1

AU2021445820A1 - CELL STRAIN FOR TGFβ DETECTION AND HIGH-PRECISION TGFβ DETECTION METHOD

Info

Publication number: AU2021445820A1
Application number: AU2021445820A
Authority: AU
Inventors: Josephine IARIA; Hongjian ZHU
Original assignee: Jiangsu Huagene Medical Research Institute Co Ltd
Current assignee: Jiangsu Huagene Medical Research Institute Co Ltd
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2023-10-19
Also published as: CN117677692A; WO2022241762A1

Abstract

Provided are a cell strain for TGFβ detection and a high-precision TGFβ detection method. Specificalla y, the cell strain for TGFβ detection in human samples is created by screening human cells with a TGFβ response at least 300 times. The method can be used for detecting the human samples and is not affected by the human samples. Compared with an ELISA detection method, the sensitivity is increased 30-fold, and a TGFβ content of 1 pg/ml can be detected; and at the same time, the method of the invention has a high selectivity and high reliability for detecting molecules. Moreover, the high accuracy and reproducibility of the above method have been confirmed by a large number of tests carried out on a cell culture medium, mouse serum, human serum and plasma.

Description

Description CELL STRAIN FOR TGFp DETECTION AND HIGH-PRECISION TGFp DETECTION METHOD

Technical field

The present invention relates to a cell line for detection of TGFp(Transforming Growth Factor Beta) and a TGFP detection method with high precision. The present invention can be used in the early aided detection and prevention of autoimmune diseases, aging, and cancer, and belongs to the technical field of biotechnology.

Background Art

Autoimmune diseases are a group of disorders which causes damage to body tissues due to wrong immune responses of human body's immune system against its own antigens. According to their pathology and symptoms, they are mainly classified into two categories: organ-specific diseases and systemic diseases, and are characterized by local or systemic inflammation, enlarged lymph nodes, and decreased leukocytes in the blood. Common autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis.

Aging refers to the intrinsic degradation process of the human body, which can be categorized into two categories: physiological aging and pathological aging. It primarily involves decrease in cellular metabolism, change of tissue structure, and gradual loss of physiological functions. The specific manifestations of aging include cardiovascular systemic sclerosis, decreased nerve conduction velocity, and weakened digestive and respiratory functions.

Cancer refers to abnormal cell proliferation, invasion of surrounding tissues, and whole body diffusion, all stemming from abnormal cell proliferation caused by multiple genetic mutations. Early clinical symptoms of cancer typically include tumor growth or localized pain, but sometimes there is no apparent symptom. In the advanced stage, cancer cells spread through the bloodstream and lymphatic system, adding difficulty to treatment, and at the same time resulting in a very high mortality rate.

Existing methods for detecting autoimmune diseases, aging, and cancer mainly rely on Enzyme Linked Immunosorbent Assay (ELISA), which detects molecular indexes in the blood to judge the condition. However, ELISA technology has drawbacks of low reproducibility, low sensitivity, low selectivity and high detection cost, so has significant limitations for early disease detection. The cost of a single ELISA test exceeds $10, its sensitivity is around 30 pg/ml, and its specificity is relatively poor.

Furthermore, current researches show that, for autoimmune diseases, aging, and cancer, the content of Transforming Growth Factor Beta (TGFI) in the serum changes greatly with the progression of diseases. In comparison to healthy individuals, mice and human patients with lupus erythematosus have lower contents of active TGF in their serum. Middle-aged mice displaying signs of aging have higher contents of active TGFP in their serum compared to young mice. Moreover, mice receiving TGF1p-inhibitory vaccines experience slower tumor growth rates

Description compared to the mice not receiving the vaccine. Therefore, TGF$ can serve as an effective prediction and diagnosis molecule for early detection of autoimmune diseases, aging, and cancer.

The methods disclosed in "New reagents for improved in vitro and in vivo examination of TGF-$ signaling" (Growth Factors, October 2011; 29(5): 211-218) are not suitable for detecting human samples, as their detection sensitivity is significantly diminished when the method is applied to human specimens.

Through technological and application innovations, the present invention addresses the problems of existing ELISA technology. The provided TGFP detection method can be used for human sample detection and have enhanced sensitivity, and provide more efficient early detection and prevention method for autoimmune diseases, aging, and cancer.

One of the objectives of this invention is to provide a cell line for TGFp (Transforming Growth Factor Beta) detection. This cell line, which is created after screening, is a human derived cell line with a TGFP response that is 300 fold or higher, or a human-derived cell line with a detection sensitivity of 1 pg/ml.

In a specific example, the cell line for TGF detection in human samples was deposited on March 10, 2021, at the Lady Mary Fairfax CellBank Australia (Address: Australia), with deposit number CBA20210033.

The second objective of this invention is to provide a cell line for TGF detection in human samples. This cell line is created by transfecting the cell line described in this invention with a reporter virus containing the reporter gene pCAGA(n)-luc.

The third objective of this invention is to provide use of the cell line mentioned in this invention or the described TGF detection cell line in TGFP detection of human samples.

The fourth objective of this invention is to provide a method for detecting TGF pin human samples. This method involves transfecting the cell line mentioned in the present invention with a reporter virus containing the reporter gene pCAGA(n)-luc, as well as co-culturing the transfected cells with the sample to detect the TGFp content in the sample.

The reporter gene pCAGA(n)-luc, as described in this invention, is a reporter gene associated with TGFP that has been previously reported in the prior art, such as the Ad-CAGA12-luc reporter used in the paper "Live Cell Imaging of the TGF-$/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System", wherein "luc" refers to luciferase (luc), which is a commonly used luciferase in this field, such as the luciferase with NCBI No. 249591.

The construction of the reporter virus containing the reporter gene pCAGA(n)-luc, as described in this invention, can be carried out using conventional method in the art, namely, transfecting the reporter gene pCAGA(n)-luc into the cell line described in this invention can also be accomplished using conventional method in the art. For example, the reporter gene pCAGA(n)-luc is cloned to an adenovirus expression vector, which is transfected into the cell

Description line after restriction enzyme digestion, then the reporter virus is extracted by lysing the cells, and subsequently the reporter virus containing pCAGA(n)-luc is transfected into the human cell line described in the present invention.

In some examples of the present invention, the co-culturing of transfected cells with samples for the detection of TGFP content in the sample can be conducted using common method in the art, such as the method disclosed in "New reagents for improved in vitro and in vivo examination of TGF-P signaling" (Growth Factors, October 2011; 29(5): 211-218). In one example, the detailed scheme is as follows:

1. Transfected cells are used to respectively detect TGFP with specified concentration and TGFI in the sample.

2. Based on the detection results of TGFP with specified concentration, a standard curve is constructed, and this curve is used to estimate the content of active TGFP in the sample.

In one example of the present invention, the samples mentioned in the present invention are human body fluids, which may comprise intracellular or extracellular fluids. For example, the samples include, but are not limited to, human serum samples.

In one example of this invention, the cell density of the cell line for detection ranges from 10 to 100 cells/pl of the sample.

The cell line, detection cell line, or TGFP detection method described in this invention find application in the preparation of aided diagnostic reagent for cancer, aging, and autoimmune diseases.

It is important to note that the specific conditions described here are just provided for illustrating the scheme of the present invention and are not intended to limit the present invention.

Experiments show that the method described in this invention can be used for detecting human samples and is not influenced by human samples. It exhibits a 30-fold increase in sensitivity compared to ELISA detection method and can detect TGF$ content as low as 1 pg/ml (as shown in Figures 4 and 5 of this invention). Additionally, it has high selectivity for detection molecules and has remarkable reliability. Through extensive tests with cell culture media, mouse serum, human serum, and plasma, the high precision and reproducibility of the present invention have been proved.

This method employs 96- or 384-well plates and can complete a detection cycle within 24 hours, significantly enhancing efficiency in terms of sample quantity and time. Moreover, the cost of a single test is less than $1. This method reduces both time and cost compared to ELISA technology. Since TGFI expression varies in autoimmune diseases, aging, and cancer, this method can be applied for the aided diagnosis of autoimmune diseases, aging, and cancer.

Description of Drawings

Description Figure 1: Comparison of contents of active TGF$ in the serum of healthy mice and mice with autoimmune disease.

Figure 2: Comparison of contents of active TGF pin the serum of young (10 weeks) and adult (40 weeks) mice.

Figure 3: Comparison of contents of active TGFp in the serum of mice inoculated with TGFp inhibitory vaccine and mice not inoculated with TGFP inhibitory vaccine.

Figure 4: Standard curves constructed from different TGF$ contents and cellular responses detected through this invention (left) and ELISA (right).

Figure 5: Comparison of TGF contents in different human serum samples detected through this invention (C) and ELISA (B).

Figure 6: Standard curves constructed from different TGF$ contents and cellular responses detected in the comparison example.

Embodiments

Specific examples are presented below to further illustrate the present invention. These examples are only provided for illustration and understanding of the invention and not for limiting the invention.

In the following examples, the cell lines transfected with the reporter virus containing pCAGA(n) luc were deposited on March 10, 2021, at the Lady Mary Fairfax CellBank Australia (Address: Australia), under deposit number CBA20210033.

Example 1: Construction of TGF$ Detection Cell Line

1. Clone the reporter gene pCAGA(n)-luc into the entry vector pENTRIA.

2. Recombine the reporter gene into the adenovirus expression vector pAd/PL-DEST using specific site attL-attR.

3. Digest the adenovirus expression vector pAd/PL-DEST with the restriction enzyme Pac.

4. Transfect the linear pAd/PL-DEST into the 293A cell line.

5. Lyse the 293A cells to extract the reporter virus.

6. Transfect the reporter virus containing pCAGA(n)-luc into the human cell line described in this invention (deposit number CBA20210033) with a multiplicity of infection (MOI) of 200 to obtain detection cell line, which is the same as the one deposited in this invention, and can have different response to various TGFp contents, as is specifically expressed in gene transcription and protein production guided by Smad3 with phosphorylation modification.

Description Example 2: Detection of Different Concentrations of TGF$ and Construction of a Standard Curve

1. Add different concentrations of TGFP to DMEM culture medium. The concentrations used in this example are 0, 0.007, 0.02, 0.06, 0.18, 0.56, 1.67, and 5 ng/ml.

2. Culture the detection cell line constructed in Example 1 in 96-well plates using the medium added with TGFp in step (1), with a density of 5x103 cells per well. After the cells reach fusion state and are cultured for 24 hours, lyse the cells using cell lysis buffer, centrifuge to separate the lysate, and extract the supernatant. Add luciferin reagent and measure the degree of fluorescence released by the reaction between luciferin and luciferase generated by reporting system in the solution using a fluorometer.

3. Based on the above detection results and the corresponding TGF concentrations, construct a standard curve as shown in Figure 4. A comparison with the traditional ELISA method reveals that the detection cell line described in this invention has an at least 300-fold TGF response. The accuracy of detecting TGFp content in serum can reach 1 pg/ml, demonstrating significant detection effect. For the same samples, when using ELISA technology, it is impossible to achieve the detection effect.

Comparison Example

Using the same method used in Example 1, a detection cell line was constructed. The difference is that the human cell line used was the human breast cancer cell MDA-MB-231, which is commercially available. The detection was performed following the same method as used in Example 2. The results, as shown in Figure 6, indicate that the cell-constructed detection cell line used in this example exhibits a minimum TGF§ response of 200-250 fold, with an accuracy of 5 pg/ml in detecting TGF$ content in serum, as is significantly lower than the results obtained in Example 2.

Example 3: Detection of Serum Samples for Autoimmune Diseases

1. For autoimmune diseases, this example starts by comparing C57BL/6-like SHIP-I-/- mice with healthy mice. SHIP-I-/- mice lack 5'-inositol triphosphate in blood cells and exhibit symptom similar to human lupus erythematosus, the specific expression of this symptom including increased bone marrow stem cell numbers, activated macrophages, increased contents of pro-inflammatory factors in serum, and excessive B cell activation. Additionally, serum samples from 60 autoimmune patients were compared with serum samples from healthy individuals (HC). The patients had systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), dermatomyositis (DM), or systemic sclerosis (SS).

2. Extract blood from mice or human, and collect the non-clotted serum portion after precipitation at 40.

3. Add 10% (v/v) serum samples obtained in step (2) to DMEM culture medium.

4. In a 96-well plate, culture the detection cell line constructed in Example 1 with the medium

Description from step (3), with a density of 5x103 cells per well. After the cells reach fusion state and are cultured for 24 hours, lyse the cells using cell lysis buffer, centrifuge to separate the lysate, and extract the supernatant. Add luciferin reagent and measure degree of fluorescence released by the reaction between luciferin and luciferase generated by reporting system in the solution using a fluorometer.

5. Based on the above detection results, mark the corresponding positions on the standard curve constructed in Example 2 for the degree of fluorescence caused by different serum samples, and calculate the TGFI content in the serum.

6. Experimental results: TGFp content in serum of mice showing symptoms of autoimmune diseases were significantly lower compared to that of healthy mice (as shown in Figure 1), and TGFI content in serum of patients with autoimmune disease were significantly lower compared to that of healthy person (as shown in Figure 5).

This example demonstrates that the cell described in this invention can be used to detect varying concentrations of TGF pand can assist prediction and diagnosis of autoimmune diseases. The detection cell line described in this invention exhibits an accuracy of1 pg/ml for detecting TGF$ content in serum, demonstrating more significant detection effect and higher sensitivity, while ELISA technology cannot achieve detection effect for the same samples (as shown in Figures 4 and 5).

Example 4: Detection of Aging Serum Samples

Regarding aging, this example compares 40-week-old and 10-week-old C57BL/6 mice.

TGF$ content detection was performed using the method described in steps (2) to (5) of Example 3. The results showed a significant increase of TGF$ content in serum in 40-week-old mice compared to 10-week-old mice (as shown in Figure 2). This indicates that it can be used for aided detection and diagnosis of aging.

Example 5: Detection of Serum Samples for Cancer

Regarding cancer, this example used C57BL/6 mice having received subcutaneous injection of B16-OVA tumor cells. Mice that received TGF$ inhibitory vaccines were compared to those that did not receive TGF$ inhibitory vaccines. For mice injected with tumor cells, significant tumors had been developed within two weeks. The TGFP inhibitory vaccine contained sT$RII-Fc, which works to block free TGF$ factors.

TGFI content detection was performed using the method described in steps (2) to (5) of Example 3. The results showed a significant decrease of TGF$ contents in serum in mice that received the TGFI inhibitory vaccine compared to mice that did not receive the TGF$ inhibitory vaccine (as shown in Figure 3). As can be seen, it can be used for the aided prediction and diagnosis of cancer. From the above results, it is evident that the method for detecting TGFp content in serum based on the standard curve has excellent performance in prediction and diagnosis of autoimmune diseases, aging, and cancer.

Claims

1. A cell line for TGFP detection of human sample, which is created by screening human-derived cells with a TGFp response that is 300 fold or higher.

2. The cell line for TGFp detection of human sample according to claim 1, characterized in that, the deposit number is CBA20210033.

3. A cell line for TGF detection of human sample, said cell line has been transfected with the reporter virus containing reporter gene pCAGA(n)-lu on the bais of the cell line of claim 1 or 2.

4. Use of the cell line of claim1 or 2 or the cell line for TGFp detection of clam 3 in TGFP detection.

5. Use of the cell line of claim 1 or 2 or the cell line for TGFP detection of clam 3 in the manufacture of medicament for aided diagnosis of cancer, aging and autoimmune diseases.

6. A method for TGFp detection of human sample, characterized in that, the reporter virus containing reporter gene pCAGA(n-lucis transfected into the cell line of claim 1 or 2, and the transfected cell is co-cultured with the human sample to detect the content of TGF in the sample.

7. The detection method of claim 5, characterized in that,

(1) the transfected cell is used to detect TGF$ of specified concentrations and TGFP in the sample;

(2) a standard curve is created based on the detection results of TGF pof specified concentrations, and is used to calculate the concentration of active TGFP in the sample.

8. The detection method of claim 5, characterized in that, the human sample is human body fluid.

9. The detection method of claim 5, characterized in that, the cell density of the transfected cells when co-cultured with the sample ranges from 10 to 100 cells/pl of the sample.

10. The detection method of claim 5, characterized in that, the method has a detection sensitivity of 1 pg/ml.