Human immortalized fallopian tube fibroblast strain as well as construction method and application thereof

Abstract

The invention discloses a human immortalized fallopian tube fibroblast strain as well as a construction method and application thereof, and belongs to the technical field of cell immortalization construction. The preparation method comprises the following steps: firstly, separating, culturing and purifying fibroblasts from human fallopian tube tissues by virtue of an enzyme digestion method, identifying markers of the fibroblasts, then introducing a simian virus 40 large T antigen gene into primary fibroblasts by virtue of a lentiviral vector, and screening by virtue of puromycin to obtain a cell strain capable of stably expressing SV40T. Finally, the human immortalized fallopian tube fibroblast which can be stably passaged is constructed. The cell strain has the characteristics of strong multiplication capacity and high hereditary stability, and the cells are not obviously aged along with the increase of the number of generations of the cells, which indicates that the human immortalized fallopian tube fibroblast line is successfully established. According to the invention, the problem that the in-vitro culture life of primary fibroblasts is limited is solved, and a reliable in-vitro cell model is provided for research in the fields of fallopian tube related diseases, ovarian cancer origin, tumor microenvironment and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell immortalization construction, and particularly relates to a human immortalized fallopian tube fibroblast cell strain and a construction method and application thereof. BACKGROUND

[0002] Ovarian cancer is the third most common malignant tumor of the female reproductive system and is currently the gynecological malignancy with the highest mortality rate. The grim clinical situation emphasizes the urgent need for research on the origin mechanism and molecular typing of ovarian cancer. According to the World Health Organization (WHO) histological classification system, epithelial tumors account for about 90% of ovarian malignancies, of which high-grade serous carcinoma (HGSC) is the most invasive major subtype. Its important pathological feature is its highly heterogeneous tumor microenvironment. Existing studies have shown that cancer-associated fibroblasts (CAFs), as the most prominent and abundant stromal cell population in the tumor microenvironment, form a dynamic interaction with a variety of stromal components (including vascular endothelial cells, immune cells, etc.) through a complex intercellular interaction network. This interaction plays a key role in mediating a series of tumor progression-related biological processes.

[0003] Existing ovarian cancer research relies on established tumor cell lines, but these cells cannot reflect the early malignant transformation process. The fallopian tube origin hypothesis suggests that high-grade serous ovarian cancer may originate from the fimbriated endometrial epithelium of the fallopian tube, and the role of fallopian tube primary fibroblasts as a key component of the microenvironment in the initiation of carcinogenesis has long been overlooked. Activation and transformation of tissue-resident normal fibroblasts are considered to be the main source of tumor-associated fibroblasts in the ovarian cancer microenvironment. Studies have shown that fallopian tube-derived fibroblasts may play a key role in the early carcinogenic process by secreting specific cytokines (such as IL-6, CXCL12) and matrix metalloproteinases (MMPs). Therefore, the establishment of a primary fallopian tube fibroblast model is of great significance for exploring the transformation mechanism of normal fibroblasts into tumor-promoting CAFs and reflecting the early occurrence of ovarian cancer. However, there is currently a lack of fallopian tube-derived fibroblast cell lines, and the in vitro proliferation capacity of primary fallopian tube fibroblasts extracted by the laboratory is limited (<10 passages), and cell senescence occurs with an increase in the number of passages, making it difficult to meet the needs of repeated experiments. Therefore, the construction of an immortalized fallopian tube fibroblast cell strain can overcome the shortcoming of the short number of passages of primary cells, while retaining the shape of fibroblasts, providing an in vitro cell model for the study of fallopian tube-related diseases and the mechanism of ovarian cancer occurrence, and has great application prospects. SUMMARY

[0004] The present application aims at the deficiencies of the prior art, and provides a human immortalized oviduct fibroblast cell strain, a construction method and application thereof.

[0005] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions. As a first aspect, the present application provides a human immortalized oviduct fibroblast cell strain, which is preserved in the China Center for Type Culture Collection, and the preservation date is June 27, 2025, the preservation number is CCTCC NO: C2025195, and the classification name is human immortalized oviduct fibroblast cell strain FT-NFs.

[0006] As a second aspect, the present application provides a construction method of the human immortalized oviduct fibroblast cell strain of the first aspect, which is constructed by infecting primary human oviduct fibroblasts with a slow virus carrying an SV40T gene, and screening a cell strain stably expressing SV40T.

[0007] Further, the construction method specifically comprises the following steps: (1) Isolating, purifying and culturing primary human normal oviduct tissue-derived fibroblasts; wherein the purification adopts a differential adhesion method.

[0008] (2) Plating: plating the primary oviduct fibroblasts in a 12-well plate, with a density of 1x10 5 -5x10 5 cells per well, preferably 5x10 5 cells per well.

[0009] (3) Slow virus infection: after 24 hours of plating, the cells are changed, and SV40T virus liquid is added for continuous culture, the titer of the SV40T virus liquid is preferably 10 8 IU / mL, the addition amount per well is 2-4 μL, preferably 3 μL. After 24 hours, the medium is supplemented for continuous culture for 48 hours, and then the medium is changed every other day for regular culture. When the cell density reaches about 80%, the cells are regularly passaged and re-infected with the virus.

[0010] (4) Puramycin screening: after several days of culture, when the cell state is stable and the cell density is about 80%, the complete medium and puramycin are added for drug screening culture for 3 days, the final concentration of puramycin in the complete medium is 1-5 μg / mL, preferably 2 μg / mL, and the cell strain stably expressing SV40T is screened out, the complete medium is changed every other day, and when the cell density reaches 80%, trypsin digestion is used for subculture.

[0011] As preferred, the step (1) of isolating the primary human oviduct fibroblast cells specifically comprises the following steps: wearing sterile gloves, cutting about 1 / 3 of the normal oviduct umbrella end tissue with a surgical blade and transferring the tissue to a sterile centrifuge tube containing D-Hanks buffer; rinsing the tissue with PBS and trimming it into 1mm 3 of tissue pieces; enzymatically digesting the tissue pieces with collagenase IV and collagenase I at 37℃ and 200rpm for 30 minutes; after enzymatic digestion, filtering the cell suspension through a 70μm cell filter, centrifuging; taking the cell precipitate, lysing the red blood cells; after lysis termination, centrifuging; taking the cell precipitate, resuspending it with a special complete culture medium, and uniformly spreading it in a culture dish; placing the culture dish in a constant temperature incubator at 37℃ and 5% CO2, and performing adherent culture; and changing the liquid the next day.

[0012] Further, the components of the special culture medium include: 10% (v / v) FBS, 1% of 100x penicillin-streptomycin mixture, DMEM / F12 culture medium, and 50ng / mL human fibroblast growth factor.

[0013] As preferred, the step (1) of purification specifically comprises the following steps: routinely culturing the isolated primary cells, discarding the culture medium after more cells adhere, washing with PBS, trypsinizing until more cells are observed to be rounded, blowing down the cells, collecting and centrifuging; resuspending the cell precipitate and inoculating for in vitro culture, after 1h, gently aspirating the culture medium to remove the non-adherent cells, washing with PBS, and replacing the fresh complete culture medium for culture; and using differential adhesion to purify and subculture the primary oviduct cells for 2 times.

[0014] As preferred, after the step (1) of purification, the markers of the human primary oviduct fibroblast cells can be detected by immunofluorescence to identify the cell type.

[0015] As preferred, the step (3) of adding the SV40T virus liquid further comprises: adding a virus infection enhancer polybrene (polybrene) to make the final concentration 4-10μg / mL, preferably 6μg / mL.

[0016] As preferred, the step (3) of virus infection uses a culture medium volume of 1mL, and the next day the culture medium is supplemented to a volume of 1mL.

[0017] As a third aspect, the present application provides the application of the cell strain of the first aspect and the cell strain constructed by the construction method of the second aspect in the research of oviduct-related diseases and / or ovarian cancer occurrence and cell communication in the microenvironment, wherein the cell strain serves as an in vitro cell model.

[0018] The beneficial effects of the present application are: the present application purifies and cultures primary fibroblasts from normal fallopian tube tissue specimens, and uses a lentivirus vector to introduce an SV40T gene into the primary fibroblasts, thereby constructing an immortalized human fallopian tube fibroblast strain, providing a stable in vitro cell model for studying the transformation mechanism of normal fibroblasts into CAFs and a co-culture system basis; promoting the study of the ovarian cancer microenvironment, and helping to further explore the early malignant transformation process of ovarian cancer. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a cell morphology chart of human immortalized fallopian tube fibroblasts under a microscope; wherein, Figure 1 A in Figure 1 is a morphology chart of non-immortalized human fallopian tube fibroblasts under magnifications of 5x, 10x and 20x, Figure 1 B in Figure 1 is a morphology chart of human immortalized fallopian tube fibroblasts under magnifications of 5x, 10x and 20x; Figure 2 Figure 2 is a result chart of immunofluorescence identification of non-immortalized and immortalized human fallopian tube fibroblasts; wherein, Figure 2 A in Figure 2 is an immunofluorescence staining result chart of α-SMA, Figure 2 B in Figure 2 is an immunofluorescence staining result chart of vimentin, Figure 2 C in Figure 2 is an immunofluorescence staining result chart of CK7. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application, and then the present application will be further described. Unless specifically stated, the technical means used in the present application are methods well known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present application, and the essence and scope of the present application are limited only by the claims. All other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0021] The source of the specimen is as follows: on March 2025, a specimen was taken from a 51-year-old patient with adenomyosis after surgery in the Department of Gynecology and Obstetrics of Zhejiang University Hospital, and the patient signed the informed consent form of the biological sample resource center of the Department of Gynecology and Obstetrics of Zhejiang University Hospital before the operation. After the specimen was removed on the operating table, 1 / 3 volume of the fallopian tube umbrella end tissue was placed in D-Hanks buffer and taken back to the laboratory as soon as possible for operation in a clean bench. If a certain period of time is needed for subsequent operation, it is placed in DMEM / F12 culture medium (VivaCell, China, C3132-0500) containing 10% (v / v) FBS (Cellmax, China, SA101.01).

[0022] Example 1: Isolation, purification and culture of primary fallopian tube fibroblasts.

[0023] 1. In a clean bench, remove fresh fallopian tube tissue from a 15 mL centrifuge tube containing D-Hanks buffer and rinse the tissue three times with sterile PBS buffer.

[0024] 2. Use tissue scissors to trim the specimen, removing surface bleeding and necrotic tissue, and then cut the tissue into pieces of about 1mm. 3 The tissue blocks were then resuspended in DMEM / F12 medium containing 125 U / mL collagenase IV (Sigma) and 25 U / mL collagenase I (Sigma) and placed in 15 mL centrifuge tubes. The centrifuge tubes were then placed in a constant temperature shaker and the tissue blocks were enzymatically digested at 37°C and 200 rpm for 30 minutes.

[0025] 3. After enzymatic hydrolysis, add an equal volume of DMEM / F12 medium containing 10% (v / v) FBS to the centrifuge tube, filter through a 70μm cell filter, and centrifuge at 300g for 5 minutes.

[0026] 4. Resuspend the cell pellet in 10 times its volume of red blood cell lysis buffer for 3 minutes to lyse the red blood cells.

[0027] 5. Add an equal volume of DMEM / F12 medium containing 10% (v / v) FBS to the red blood cell lysis buffer from step 4, and centrifuge at 300g for 5 minutes.

[0028] 6. Discard the supernatant and resuspend the cell pellet in 10 mL of dedicated complete culture medium (the culture medium components include: 10% FBS, 1% 100× penicillin-streptomycin solution, DMEM / F12 medium, and 50 ng / mL human fibroblast growth factor), and spread it evenly in a 10 cm culture dish.

[0029] 7. Place the petri dishes in a constant temperature incubator at 37℃ and 5% CO2 for adherent culture, and change the medium the next day.

[0030] 8. Culture according to standard methods until cells are visible adhering and growing in the culture dish. Since primary human fallopian tube fibroblasts adhere more easily and are easier to digest than other cell types, when the cell density reaches 75%, fibroblast purification is performed using differential adhesion. The procedure is as follows: (8.1) Discard the culture medium, wash the cells twice with PBS, add 1 mL trypsin, digest at 37°C, and observe under a microscope. When there are many rounded cells, stop the digestion with 2 mL complete culture medium. The remaining undigested adherent cells are other miscellaneous cells. Gently collect the digested cells in a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min.

[0031] (8.2) Resuspend the cell pellet and inoculate a culture dish, add complete culture medium to cover the dish, and place the culture dish in a 37°C, 5% CO2 incubator.

[0032] (8.3) After 1 h, gently aspirate the culture medium to remove the non-adherent cells, wash twice with PBS, and add fresh complete culture medium to the original culture dish for culture.

[0033] (8.4) The primary cells from the fallopian tube tissue are passaged twice using the differential adhesion method to improve the purity of the fibroblasts. It can be observed that the fibroblasts are spindle-shaped, irregular star-shaped, and significantly stretched after adhesion. Further, the primary fallopian tube fibroblasts can be identified using immunofluorescence technology.

[0034] Example 2: Process of immortalization of primary fallopian tube fibroblasts.

[0035] 1. Plating: Plate the primary fallopian tube fibroblasts obtained after isolation and purification in Example 1 at a density of 5 x 10 5 cells per well in a 12-well plate.

[0036] 2. Lentivirus infection: Change the medium 24 h after plating, add 1 mL of DMEM / F12 complete culture medium to each well, and add 3 μL of simian virus 40 large T antigen (SV40T) virus solution (Aibimo Biotechnology Co., Ltd., virus titer: 10 8 IU / mL) and virus infection enhancer polybrene (final concentration 6 μg / mL) for culture. After 24 h, supplement 1 mL of complete culture medium for continuous culture for 48 h, and then change the medium every other day for regular culture.

[0037] 3. When the cell density reaches 80%, the cells are passaged at 1:2 for culture. Repeat step 2 of this example for virus re-infection to improve the infection efficiency.

[0038] 4. Puromycin Screening: After several days of culture, when the cells are stable and the cell density is around 80%, add 1 mL of complete medium containing 2 μg / mL puromycin for drug screening. Culture for 3 days to screen for cell lines that stably express SV40T. Change the medium with complete medium every other day. When the cell density reaches 80%, digest with trypsin and continue subculturing to obtain an immortalized human fallopian tube fibroblast.

[0039] Example 3: Identification of a human immortalized fallopian tube fibroblast.

[0040] 1. Morphological observation of immortalized human fallopian tube fibroblasts: Human immortalized fallopian tube fibroblasts were passaged and cultured. The growth of these cells compared to unimmortified live cells was observed under a light microscope (5×, 10×, 20×). Results are shown below. Figure 1 , Figure 1 In this context, A represents a non-immortalized human fallopian tube fibroblast. Figure 1 B in the image represents human immortalized fallopian tube fibroblasts after the 8th passage under an optical microscope. It can be observed that after immortalization, the cells in this cell line grow vigorously, the background is clear, there are few impurities, the adherent cells are fibrous, the morphology tends to be homogeneous, and no senescence phenotype is observed.

[0041] 2. Identification by fluorescent staining of specific proteins in immortalized fallopian tube fibroblasts: Primary immortalized fallopian tube fibroblasts were digested with trypsin and seeded into 12-well plates containing pre-placed cell spreaders at a density of 3 × 10⁶ cells / well. 5 Cells were cultured in each well for 48 hours. The culture medium was discarded, and cells were washed twice with PBS. They were then fixed with 4% paraformaldehyde for 30 minutes, followed by washing three times with PBS for 5 minutes each time. Cells were then permeated with PBS containing 0.1% Triton X-100 for 10 minutes, followed by washing three times with PBS for 5 minutes each time. Afterward, cells were blocked with 3% BSA for 30 minutes. The BSA was discarded, and four groups were set up, each containing primary antibodies against anti-α-SMA, anti-Fap, anti-vimentin, and anti-CK7, respectively. Cells were incubated overnight at 4°C on a shaker. The next day, the primary antibodies were washed away with PBS, and the cells were incubated with the corresponding fluorescent secondary antibodies at room temperature in the dark for 1 hour. The secondary antibodies were then washed away with PBS. Slides were mounted with mounting medium containing DAPI and observed and photographed under a confocal microscope. Results are shown below. Figure 2 Immortalized fallopian tube fibroblasts expressed α-SMA and vimentin (…). Figure 2 A and B in the text), but do not express the epithelial cell-specific marker CK7 ( Figure 2 (C in the text) above clarifies the establishment of immortalized fallopian tube fibroblasts.

Claims

1. A human immortalized fallopian tube fibroblast cell line, characterized in that, It is deposited at the China Center for Type Culture Collection on June 27, 2025, with accession number CCTCC NO: C2025195.

2. A method for constructing a human immortalized fallopian tube fibroblast cell line as described in claim 1, characterized in that, The process includes the following steps: infecting primary human fallopian tube fibroblasts with lentiviruses carrying the SV40T gene, and screening for cell lines that stably express SV40T.

3. The construction method according to claim 2, characterized in that, Prior to infection with a lentivirus carrying the SV40T gene, the primary human fallopian tube fibroblasts were isolated and purified using a differential adhesion method.

4. The construction method according to claim 2, characterized in that, The method of infecting primary human fallopian tube fibroblasts with a lentivirus carrying the SV40T gene includes the following steps: primary human fallopian tube fibroblasts are cultured at a density of 1 × 10⁻⁶ cells per well. 5 -5×10 5 Plating cells at a density of 1000 cells / well, adding 2-4 μL of 10 ... 8 IU / mL of SV40T virus solution was mixed with a virus infection enhancer to achieve a final concentration of 4-10 μg / mL.

5. The construction method according to claim 2, characterized in that, The cell lines that stably express SV40T were selected using puromycin at a final concentration of 1-5 μg / mL.

6. The construction method according to claim 2, characterized in that, After obtaining a cell line that stably expresses SV40T, the process also includes: trypsin digestion followed by passage culture.

7. The application of the human immortalized fallopian tube fibroblast line according to claim 1 in research on fallopian tube-related diseases and / or ovarian cancer, characterized in that, The human immortalized fallopian tube fibroblast cell line was used as an in vitro cell model.

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