Construction and identification of immortalized human endometriosis eutopic stromal cells

An endometrial and heterotopia technology, applied in reproductive tract cells, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of less attention to endometrial cells, and achieve cell growth. The effect of extended cycle, long life cycle and high state activity

Inactive Publication Date: 2019-11-08
THE FIRST AFFILIATED HOSPITAL OF XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing patents only focus on the endometrial culture of ...

Method used

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  • Construction and identification of immortalized human endometriosis eutopic stromal cells
  • Construction and identification of immortalized human endometriosis eutopic stromal cells
  • Construction and identification of immortalized human endometriosis eutopic stromal cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: lentivirus infection of mesenchymal cells in situ to construct iheESCs cells

[0047] Transfer the human hTERT gene to the GV492 vector, mix the transfection reagent Lipo2000 and the lentiviral packaging plasmid VSVG / PMDL / REV, add it to the 293T tool cells, collect the virus supernatant after 24 hours and centrifuge to remove impurities; the supernatant is incubated in uterus The eutopic mesenchymal cells of a patient with membranous dysplasia were replaced with fresh culture medium after 24 hours. The red transfection fluorescence of the cells was observed under a fluorescent microscope, and the expression level of hTERT was significantly increased by qRT-PCR and Western Blot, indicating that the hTERT transfection was successful. Continuously cultured cells, iheESCs cells can be passed down to 35 passages, while ordinary cells can only be passed down to 18 passages, indicating that the proliferation cycle of iheESCs cells is significantly prolonged after con...

Embodiment 2

[0049] Example 2: Detection of iheESCs cell growth cycle, migration and invasion, expression of estrogen and progesterone receptors and epithelial-mesenchymal characteristic proteins

[0050] Further in-depth examination of the physiological function changes of iheESCs cells. CCK-8 assay was used to detect the growth law of iheESCs cells, and it was found that there was no significant change between iheESCs cells and control cells; Western Blot was used to detect ERα, ERβ and PR and other estrogen and progesterone, epithelial-mesenchymal marker proteins Keratin, E-cadherin, Vimentin and N-cadherin and others had no significant difference; the epithelial-mesenchymal state of iheESCs cells remained unchanged through immunohistochemical experiments. The above shows that the basic characteristics of iheESCs cells have not changed significantly.

[0051] The result is as Figure 6 As shown, there was no significant difference between the growth curves of iheESCs cells and control...

Embodiment 3

[0054] Example 3: Characteristic reaction of iheESCs decidualization

[0055] The endometrium has the function of receiving fertilized eggs, and endometrial stromal cells will have important changes in decidualization during the receiving process, so the present invention focuses on verifying the decidualization ability of iheESCs cells. 10nM estrogen, 0.1 μM MPA and 0.01M cAMP dissolved in DMEM / F12 medium were used as decidualization induction medium, and iheESCs cells were cultured for 72 hours. It was found that estrogen induced the cells to become spindle-shaped, while decidual fluid significantly induced the cells to become round, and qPCR detected that iheESCs cells expressed higher insulin-like growth factor-binding protein-1 (IGFBP1) and prolactin (PRL) The expression was significantly increased, indicating that iheESCs cells still preserved the process of decidualization, suggesting that it can be used as a model cell line for the study of endometrial receptivity.

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Abstract

The invention discloses construction and identification of immortalized human endometriosis eutopic stromal cells (iheESCs), and relates to endometriosis. The invention relates to the iheESCs construction, and the identification of characteristics of iheESCs cells as endometrial stromal cells. A cell growth cycle of the constructed iheESCs cells is obviously prolonged, a proliferation rate, an estrogen/progesterone receptor, an EMT marker protein and the like are not changed, meanwhile, deciduation induction reaction, estrogen reaction and inflammatory reaction are changed, and the constructedcells do not have tumorigenicity. Therefore, compared with common primary cells, the constructed iheESCs have the characteristics of long growth cycle, stable phenotype and higher state activity, canbe suitable for research on endometriosis pathological mechanisms, endometrial receptive physiological mechanisms and the like, and have certain applicability in clinical experiments, medical biological experiments and the like.

Description

technical field [0001] The present invention relates to endometriosis, in particular to the construction and identification of immortalized human endometriosis eutopic stromal cells (immortalized humanendometriosis utopicstromal cells, iheESCs). Background technique [0002] Endometriosis (EMs), referred to as endometriosis, is a chronic gynecological disease characterized by estrogen dependence and inflammatory response, often accompanied by pelvic pain, abnormal menstruation, painful sexual intercourse, etc., and even infertility It seriously affects women's physical and mental health, and brings financial and spiritual burdens to families. Endometriosis refers to benign lesions in which endometrium with growth function grows and reproduces in other parts outside the uterine cavity, such as ovary and abdominal wall. The incidence rate is as high as 6% to 15%, and the recurrence rate is as high as 6% to 67%. . The pathogenesis of endometriosis involves many influencing fa...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12Q1/02
CPCC12N5/0682C12N9/1276C12N15/86C12N2510/04C12N2740/15043C12Y207/07049G01N33/5005
Inventor 陈琼华黄志雄林佳慧程蕾
Owner THE FIRST AFFILIATED HOSPITAL OF XIAMEN UNIV
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