CN106011064A - Method for establishing human normal prostate epithelial neuroendocrine subtype cell line - Google Patents
Method for establishing human normal prostate epithelial neuroendocrine subtype cell line Download PDFInfo
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- CN106011064A CN106011064A CN201610385303.9A CN201610385303A CN106011064A CN 106011064 A CN106011064 A CN 106011064A CN 201610385303 A CN201610385303 A CN 201610385303A CN 106011064 A CN106011064 A CN 106011064A
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Abstract
The invention discloses a method for establishing a human normal prostate epithelial neuroendocrine subtype cell line. The method includes culture of an NHPrE1 cell line, separation and purification of neuroendocrine subtype NTU2 cells, NTU2 cell proliferation, NTU2 cell cloning, in vitro identification of NTU2 cell and in vivo identification of NTU2 cell. The establishment of the cell line (NTU2) provides basis experimental materials for the studies of prostate neuroendocrine cells (NEC) function, oncobiological effect of neuroendocrine cells in occurrence and development of prostate cancer and interaction of neuroendocrine cells with tumor cell molecules, and the screening, development and research on drugs for hormone-independent prostate cancer (CRPC).
Description
Technical field
The present invention relates to the method for building up of a kind of people normal prostatic epithelium neuroendocrine sub-types of cells system.
Background technology
Neuroendocrine cell (NEC) is the terminally differentiated cells generally acknowledged at present, is cell type common in the tumors such as pulmonary carcinoma, gastrointestinal cancer and carcinoma of prostate.There is the feature of anti-Radiotherapy chemotherapy.Its terminal differentiation characteristic constrains carrying out of the applied researcies such as its basic research in vitro and new medicament screen.
Summary of the invention
The research of the anti-Radiotherapy chemotherapy of carcinoma of prostate that after it is an object of the invention to go hormone therapy for carcinoma of prostate, the neuroendocrine differentiated of prostate cancer tissue inner cell causes, and the research such as the screening of relevant new drug, exploitation, it is provided that the method for building up of the people normal prostatic epithelium neuroendocrine sub-types of cells system of infrastest material.
The technical solution of the present invention is:
(this cell line is the cell line that applicant sets up, and is abbreviated as in a kind of people normal prostatic epithelium neuroendocrine sub-types of cells systemNTU2) method for building up, it is characterized in that: comprise the following steps:
(1) cultivation of NHPrE1 cell line: 1:1 DMEM/F12, adds 5% FBS liquid, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within 2-3 days, changing liquid once, 1:3-4 passes on；
(2) neuroendocrine sub-types of cells NTU2 separates: after NHPrE1 cell culture trypsinization, 1000 RPM centrifugal collecting cells, after 1X PBS washs three times, closes 20 minutes in 4 DEG C with 3% BSA；Add 1:50 mouse-anti people ChG A antibody (sigma), after 4 DEG C hatch 30 minutes, wash three times with 1X PBS；Add sheep anti mouse magnetic bead antibody, after 4 DEG C hatch 30 minutes, carry out cell separation with mini MACS Separator；
(3) amplification of NTU2 cell: by separating obtained positive cell 1:1 DMEM/F12 culture fluid, adds 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within every 3 days, change liquid once；
(4) NTU2 Cell-cloned: NTU2 cell 1:1 DMEM/F12 culture fluid will be expanded, add 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；It is seeded to 96 orifice plates by 0.5 cells/well；Within every 3 days, change liquid once, proceed to culture bottle after growing into single cell clone and expand by above-mentioned culture medium；
(5) NTU2 cells in vitro is identified: 14mm round coverslip poly-D-lysine puts into 24 orifice plates after being coated, inoculation NTU2 cell, after growing to 60-70% density, paraformaldehyde is fixed, after PBS fully washs, carry out immunocytochemistry analysis with neuroendocrine cell marker molecule antibody ChG A and Syn respectively；
(6) identify in NTU2 cyton: NTU2 cell by 20% ratio respectively with PC-3 and DU145 mixing with cells after, application organizes recombination method to plant respectively to nude mice subrenal capsule；Taking out tumor body after becoming tumor, paraformaldehyde is fixed, and paraffin embedding is cut into slices, and carries out Fluorescent immunohistochemistry analysis with neuroendocrine marker molecule.
NTU2 cell line of the present invention is for the functional study of prostate neuroendocrine cell (NEC).Occur and oncobiology effect and the research of molecular action mutual with tumor cell thereof in development in carcinoma of prostate for neuroendocrine cell.New medicament screen developmental research for hormone independent prostate cancer (CRPC).
The functional study of prostate neuroendocrine cell (NEC), the neuroendocrine cell new medicament screen developmental research of oncobiology effect and the research of molecular action mutual with tumor cell and hormone independent prostate cancer (CRPC) in carcinoma of prostate occurs and develops that is established as of this cell line of the present invention (NTU2) provides infrastest material.
Below in conjunction with embodiment, the invention will be further described.
Detailed description of the invention
The method for building up of a kind of people's normal prostatic epithelium neuroendocrine sub-types of cells system (NTU2), comprises the following steps:
(1) cultivation of NHPrE1 cell line: 1:1 DMEM/F12, adds 5% FBS liquid, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within 2-3 days, changing liquid once, 1:3-4 passes on；
(2) neuroendocrine sub-types of cells NTU2 separates: after NHPrE1 cell culture trypsinization, 1000 RPM centrifugal collecting cells, after 1X PBS washs three times, closes 20 minutes in 4 DEG C with 3% BSA；Add 1:50 mouse-anti people ChG A antibody sigma, after 4 DEG C hatch 30 minutes, wash three times with 1X PBS；Add sheep anti mouse magnetic bead antibody, after 4 DEG C hatch 30 minutes, carry out cell separation with miniMACS Separator；
(3) amplification of NTU2 cell: by separating obtained positive cell 1:1 DMEM/F12 culture fluid, adds 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within every 3 days, change liquid once；
(4) NTU2 Cell-cloned: NTU2 cell 1:1 DMEM/F12 culture fluid will be expanded, add 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；It is seeded to 96 orifice plates by 0.5 cells/well；Within every 3 days, change liquid once, proceed to culture bottle after growing into single cell clone and expand by above-mentioned culture medium；
(5) NTU2 cells in vitro is identified:
a.
Cultivating cell to fix through glutaraldehyde, after 2% Agarose embedding, glutaraldehyde carries out electron microscopyc sample preparation observation after fixing again.
b.
14mm round coverslip poly-D-lysine puts into 24 orifice plates after being coated, inoculation NTU2 cell, after growing to 60-70% density, paraformaldehyde is fixed, after 1X PBS fully washs, carry out immunocytochemistry analysis with neuroendocrine cell marker molecule antibody ChG A and Syn respectively；
(6) identify in NTU2 cyton: NTU2 cell by 20% ratio respectively with PC-3 and DU145 mixing with cells after, application organizes recombination method to plant respectively to nude mice subrenal capsule；Taking out tumor body after becoming tumor, paraformaldehyde is fixed, and paraffin embedding is cut into slices, and carries out immunohistochemical analysis with neuroendocrine marker molecule.
Cell is built based material and is originated: immortal human normal prostatic epithelium progenitor cell line (NHPrE1).This cell line is awarded to build by river penetrating judgment.And proceeded to GFP as molecular marker.
Claims (3)
1. a method for building up for people's normal prostatic epithelium neuroendocrine sub-types of cells system, is characterized in that: comprise the following steps:
(1) cultivation of people's normal prostatic epithelium progenitor cell line NHPrE1 cell line: 1:1
DMEM/F12, adds 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within 2-3 days, changing liquid once, 1:3-4 passes on；
(2) separation of people's normal prostatic epithelium neuroendocrine sub-types of cells system NTU2 and purification: after NHPrE1 cultivates cell trypsin enzymic digestion, 1000 RPM centrifugal collecting cells, after 1 × PBS washs three times, with 3% BSA in 4 DEG C of closings 20 minutes；Add 1:50 mouse-anti people's ChG A antibody, after 4 DEG C hatch 30 minutes, wash three times with 1 × PBS；Add sheep anti mouse magnetic bead antibody, after 4 DEG C hatch 30 minutes, carry out cell separation with mini MACS Separator；
(3) amplification of NTU2 cell: by separating obtained positive cell 1:1 DMEM/F12 culture fluid, adds 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；In 5% CO2,37 DEG C of cultivations；Within every 3 days, change liquid once；
(4) NTU2 Cell-cloned: by amplification NTU2 cell 1:1
DMEM/F12 culture fluid, adds 5% FBS, 1% ITS, 5ng/mL EGF, 50ng/mL BPE；It is seeded to 96 orifice plates by 0.5 cells/well；Within every 3 days, change liquid once, proceed to culture bottle after growing into single cell clone and expand by above-mentioned culture medium.
The method for building up of people the most according to claim 1 normal prostatic epithelium neuroendocrine sub-types of cells system, it is characterized in that: also carry out NTU2 cells in vitro qualification after step (4): 14 mm round coverslip poly-D-lysine puts into 24 orifice plates after being coated, inoculation NTU2 cell, after growing to 60-70% density, paraformaldehyde is fixed, after 1 × PBS fully washs, carry out immunocytochemistry analysis with neuroendocrine cell marker molecule antibody Chromogranin A and Synapsin respectively.
The method for building up of people the most according to claim 1 normal prostatic epithelium neuroendocrine sub-types of cells system, it is characterized in that: also carry out after step (4) identifying in NTU2 cyton: NTU2 cell by 20% ratio respectively with people Advanced prostate cancer PC-3 and DU145 mixing with cells after, application organizes recombination method to plant respectively to nude mice subrenal capsule；Taking out tumor body after becoming tumor, paraformaldehyde is fixed, and paraffin embedding is cut into slices, and carries out immunohistochemical analysis with neuroendocrine marker molecule.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237535A (en) * | 2014-09-30 | 2014-12-24 | 天津市泌尿外科研究所 | Application of neurotensin in diagnosis on castration resistant prostate cancer neuroendocrine subtype and prognosis judgment |
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- 2016-06-03 CN CN201610385303.9A patent/CN106011064A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237535A (en) * | 2014-09-30 | 2014-12-24 | 天津市泌尿外科研究所 | Application of neurotensin in diagnosis on castration resistant prostate cancer neuroendocrine subtype and prognosis judgment |
Non-Patent Citations (2)
| Title |
|---|
| JIANG M ET AL: "Functional remodeling of benign human prostatic tissues in vivo by spontaneously immortalized progenitor and intermediate cells", 《STEM CELLS》 * |
| 刘欣等: "前列腺癌干细胞的研究进展", 《中国男科学杂质》 * |
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