CN107034190B

CN107034190B - Human placenta position trophoblastic tumor cell line

Info

Publication number: CN107034190B
Application number: CN201610080657.2A
Authority: CN
Inventors: 康玉; 刘齐雨; 李可; 卢冲; 蒲萄; 张明星; 刘海鸥; 罗建民; 李桂玲; 徐丛剑
Original assignee: Fudan University Shanghai Cancer Center; Obstetrics and Gynecology Hospital of Fudan University
Current assignee: Fudan University Shanghai Cancer Center; Obstetrics and Gynecology Hospital of Fudan University
Priority date: 2016-02-04
Filing date: 2016-02-04
Publication date: 2020-12-08
Anticipated expiration: 2036-02-04
Also published as: CN107034190A

Abstract

The invention belongs to the field of microbial animal cell lines, and particularly relates to a human placenta trophoblastic tumor cell line and an establishment method thereof. The invention establishes a human placenta part trophoblastic tumor cell line, named as PSTT-1 with the preservation number of 10882, through the primary culture of focus in vitro uterus of a placenta part trophoblastic tumor patient, and the biological characteristics of the invention comprise: the cell monolayer grows adherent to the wall, loses contact inhibition, has uneven shape and takes a multi-protuberant star-shaped or spindle-shaped flat structure; the in vitro culture has good growth and can be continuously and stably passed for more than 30 generations; the result of karyotyping is a normal female karyotype: 46, XX. The PSTT-1 cell line can be used for researching etiology, transfer mechanism, drug resistance mechanism and the like of the trophoblastic tumor disease at the placenta part.

Description

Human placenta position trophoblastic tumor cell line

Technical Field

The invention belongs to the field of microbial animal cell lines, and particularly relates to establishment of a human placenta trophoblastic tumor cell line and an establishment method thereof.

Background

Research reports that Placental Site Trophoblastic Tumors (PSTT) originate from the intermediate trophoblastic cells of the placenta implantation site, a special type of Gestational Trophoblastic Neoplasms (GTN); the disease is relatively rare clinically, and accounts for about 1 to 2 percent of gestational trophoblastic tumors.

Studies have shown that trophoblastic tumors in the placenta region mostly occur at the age of childhood, with irregular vaginal bleeding or menorrhagia as the main symptoms after amenorrhea. According to literature reports, the clinical course of PSTT is mostly benign, but about 10% to 15% of cases appear aggressive. Studies have shown that most lesions in this condition are confined to the uterus with a better prognosis, but once metastasis occurs, the prognosis is poor despite surgery and combination chemotherapy; unlike other types of gestational trophoblastic tumors, PSTT is not sensitive to chemotherapy, and the corresponding mechanism is not clear, so that the operation is the current clinical main treatment means, and the operation can cause the patient in the childbearing age to lose the natural pregnancy capability; therapeutic intervention regimens for preserving fertility in such patients are still under investigation in clinical practice.

Because the placental site trophoblastic tumor is rare, the current literature reports are mostly small samples or reports, the progress of related basic research is relatively slow, only 1 report about the establishment of the PSTT cell line exists at present, and 1 PSTT cell line IST-2 is successfully established in Shih, I.M. and the like in 2002; for subsequent applications of this cell line, only 1 literature report, Kobel, M. et al, applied IST-2 explored the role of the MAPK pathway in PSTT invasion and migration.

In view of the fact that the establishment of tumor cell lines plays an important role in the basic research of tumors, but no universal PSTT cell line exists worldwide, the inventors of the present application intend to establish a placenta trophoblastic tumor cell line, and further explore the etiology, metastasis mechanism, drug resistance mechanism and clinical intervention of placenta trophoblastic tumors.

The prior art related to the present invention and related thereto are:

1.Ajithkumar,T.V.,et al.,Placental site trophoblastic tumor.Obstet Gynecol Surv,2003.58(7):p.484-8.

2.Behtash,N.and M.Karimi Zarchi,Placental site trophoblastic tumor.J Cancer Res Clin Oncol,2008.134(1):p.1-6.

3.Schmid,P.,et al.,Prognostic markers and long-term outcome of placental-site trophoblastic tumours:a retrospective observational study.Lancet,2009.374(9683):p.48-55.

4.Zhao,J.,et al.,Reservation of fertility for seventeen patients with placental site trophoblastic tumor，Zhonghua Fu Chan Ke Za Zhi,2014.49(4):p.265-9.

5.Shih,I.M.,G.Singer,and R.J.Kurman,Establishment of intermediate trophoblastic cell lines from PSTT and complete hydatidiform mole.Modern Pathology,2002.15(1):p.210A-210A.

6.Kobel,M.,et al.,Activation of mitogen-activated protein kinase is required for migration and invasion of placental site trophoblastic tumor.Am J Pathol,2005.167(3):p.879-85.。

disclosure of Invention

The invention aims to provide a human placental trophoblastic tumor cell line PSTT-1 aiming at the current situation that the establishment of a PSTT cell line and related tumor cell lines which are not universal in the world at present have important roles in the research of tumors, and the cell line can be used for the research of etiology, transfer mechanism, drug resistance mechanism, clinical intervention and the like of placental trophoblastic tumor diseases.

The invention provides a human placenta trophoblastic tumor cell line, which is named as PSTT-1 and is preserved by China general microbiological culture Collection center (CGMCC) in 2015 at 6, 4 months, wherein the preservation unit address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and is deposited under No. 10882.

The human placenta trophoblastic tumor cell line is prepared through the following steps,

(I) taking materials

Obtaining an excised fresh placenta position trophoblastic tumor resection specimen, immersing focus tissues into a sterile Hanks solution for cleaning, and removing connective tissues and necrotic tissues;

(II) Primary culture

Inoculating the tumor tissue block on the bottle wall of a T25 culture bottle, and standing for culture, wherein cells grow out of the tissue block after about 3 weeks;

(III) cell purification

Inoculating for about 7 weeks, transferring to generation 1, digesting with 0.25% trypsin, inoculating the cell suspension into a culture dish, observing under an inverted phase contrast microscope to see that part of cells adhere to the wall, and pouring the cell suspension into another culture bottle for continuous culture; repeating the passage for 3 times according to the method to completely separate the tumor cells from the fibroblasts;

(IV) subculturing

When the bottom of the bottle is full of cells, sucking out the culture solution, rinsing with PBS buffer solution, digesting with 0.25% trypsin, carrying out passage according to the ratio of 1:3, and transferring to 30 generations at present;

the human placenta trophoblastic tumor cell line is obtained by biological characteristic identification and is named as PSTT-1 with the preservation number of No. 10882.

In the invention: the in vitro conventional culture condition is RPMI1640 cell culture medium containing 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% HEPES, and 1% double antibody, the culture temperature is 37 deg.C, and the gas environment is 5% CO₂And/95% air, and the humidity is saturated humidity.

The cell line PSTT-1 of the invention has the following biological properties:

1. the monolayer grows adherent to the wall, loses contact inhibition, has inhomogeneous shape, and has a multi-protuberant star-shaped or spindle-shaped flat structure;

2. the in vitro culture has good growth, the subculture is carried out once every 5 days, and the continuous and stable subculture can be carried out, and the subculture is transferred to more than 30 generations at present;

3. the result of karyotyping is a normal female karyotype: 46, XX;

the PSTT-1 is highly similar to the analysis result of the STR (short distance repeat) of the tumor tissue, the PSTT-1 immunocytochemical staining is consistent with the immunohistochemical staining result of the tumor tissue, and the PSTT-1 is proved to be derived from the tumor tissue.

The invention provides a human placenta trophoblastic tumor cell line PSTT-1 which can be used for researching etiology, transfer mechanism, drug resistance mechanism, clinical intervention and the like of placenta trophoblastic tumor diseases.

Drawings

FIG. 1, PSTT-1 cell morphology (inverted phase contrast microscope 100 ×).

FIG. 2, PSTT-1 cell growth curve.

FIG. 3, karyotyping of PSTT-1 chromosome.

FIG. 4, PSTT-1 and tumor tissue STR analysis.

FIG. 5, PSTT-1 stained with tumor tissue HE (100 ×).

FIG. 6, PSTT-1 immunocytochemical staining (100X) and tumor tissue immunohistochemical staining (100X).

Detailed Description

EXAMPLE 1 preparation of human placental site trophoblastic tumor cell line PSTT-1

(1) Material taking: fresh placenta position trophoblastic tumor resection specimen (female, 29 years old, placenta position trophoblastic tumor, pathological diagnosis: { whole uterus } uterus placenta position trophoblastic tumor) is obtained from auxiliary obstetrics and gynecology hospitals of Compound Dan university in 9 months in 2014, focus tissue is taken to be 1cm³Immersing in sterile Hanks solution for cleaning, and removing connective tissue and necrotic tissue;

(2) primary culture

The tumor tissue is cut to about 1mm³Uniformly inoculating the tissue blocks on the bottle wall of a T25 culture bottle, standing for culture, changing the liquid for 1 time in half every 3 days, and observing the growth of cells from the tissue blocks after about 3 weeks;

(3) cell purification

And (3) inoculating the cell suspension for about 7 weeks, then transferring the cell suspension to the 1 st generation, digesting the cell suspension by using 0.25% trypsin, inoculating the cell suspension to a culture dish (culture solution does not contain serum), standing for 15-20 min, observing the cell suspension under an inverted phase contrast microscope until part of cells are attached to the wall, and pouring the cell suspension into another culture bottle for continuous culture. Treating according to the above method when subculturing next time, repeating for 3 times, and completely separating tumor cells from fibroblasts;

(4) subculturing

When the bottom of the bottle is full of cells, sucking out old culture solution in the original bottle, rinsing for 1 time by using PBS buffer solution, digesting by using 0.25% trypsin, carrying out passage according to the proportion of 1:3, and transferring to 30 generations at present; the human placenta trophoblastic tumor cell line is prepared, is named as PSTT-1, is preserved by China general microbiological culture Collection center (CGMCC) in 2015 at 6-30 months, and has the preservation unit address: the microbial research institute of China academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, and is deposited under No. 10882.

The in vitro conventional culture conditions of the experiment are RPMI1640 cell culture medium containing 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% HEPES and 1% double antibody, the culture temperature is 37 ℃, and the gas environment is 5% CO₂95% of air, wherein the humidity is saturated humidity;

(5) identification of biological Properties

Cell morphology: observed under an inverted phase contrast microscope, the PSTT-1 cell monolayer grows adherent to the wall, loses contact inhibition, has a larger cell body, is in a multi-protuberant star-shaped or spindle-shaped flat structure, and has an inhomogeneous shape (shown in figure 1);

cell growth curves: taking cells in logarithmic phase, adjusting cell density to 2 x 10^3/ml, uniformly mixing, inoculating the cells in a 96-well plate, and arranging 3 multiple wells; adding CCK-8 reagent at the same time for 4 hours after inoculation for 6 days continuously, measuring the absorbance (OD) at 450nm by using a microplate reader, and drawing a cell growth curve (shown in figure 2) by taking time as a horizontal axis and an OD mean value as a vertical axis;

karyotyping analysis: taking cells in logarithmic growth phase, treating the cells with 0.25 mu g/ml colchicine for 6 hours, standing overnight at 37 ℃, collecting cells in metaphase, fixing the cells in a fixing solution (methanol: glacial acetic acid ═ 3:1), digesting the sample by trypsin, staining the cells by a Giemsa staining solution, observing and counting under a microscope, randomly counting 21 cells, wherein the number of chromosomes is 46, and selecting a well-dispersed and moderately stained division phase for karyotyping, wherein the result is that the karyotype of a normal female is: 46, XX (as shown in figure 3);

STR analysis: extracting PSTT-1 cell and tumor tissue DNA by using an Axygen kit, and carrying out PCR amplification; selecting 20 sites (including sex gene Amelogenin): D13S317, D7S820, D16S539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, PENTAD, detection of STR sites with ABI 3730XL type genetic analyzer; the results show that the tumor tissue is highly similar to the STR analysis result of the PSTT-1 cells, the detection results of the two sites of AMEL and D13S317 are different, and the rest 18 sites are completely the same, which indicates that the PSTT-1 is derived from the tumor tissue; FIG. 4 shows some of the experimental results;

the surgical specimens and monoclonal PSTT-1 cells were fixed in formalin, sectioned with paraffin embedded, and HE stained (as shown in FIG. 5); when AE1/AE3, hCG and hPL were selected to perform immunocytochemical staining on the monoclonal PSTT-1 cells, compared with immunohistochemical staining of tumor tissues (as shown in FIG. 6), the results showed that the immunocytochemical staining and immunohistochemical staining result were consistent, confirming that the differentiation phenotype of the original tumor cells was maintained.

Claims

1. A human placenta trophoblastic tumor cell line is characterized in that the cell line is a human placenta trophoblastic tumor cell line which is named as PSTT-1 and is preserved by China general microbiological culture Collection center (CGMCC) in 2015 at 6-30 days, and the preservation number is No. 10882.

2. The human placental site trophoblastic tumor cell line according to claim 1, wherein said cell line has the following biological characteristics:

the monolayer grows adherent to the wall, loses contact inhibition, has inhomogeneous shape, and has a multi-protuberant star-shaped or spindle-shaped flat structure; the in vitro culture has good growth, and can be continuously and stably passed for more than 30 generations after being passed once every 5 days; the result of karyotyping is a normal female karyotype: 46, XX; the PSTT-1 is highly similar to the STR analysis result of the tumor tissue, and the PSTT-1 immunocytochemical staining is consistent with the immunohistochemical staining result of the tumor tissue.

3. The human placental site trophoblastic tumor cell line according to claim 1, wherein said cell line is prepared by the following method, using ex vivo tumor tissue to process and subculture to create human placental site trophoblastic tumor cell line PSTT-1, comprising the steps of:

(1) material taking: obtaining an excised fresh placenta position trophoblastic tumor resection specimen, immersing focus tissues into a sterile Hanks solution for cleaning, and removing connective tissues and necrotic tissues;

(2) primary culture: inoculating the tumor tissue block on the bottle wall of a T25 culture bottle, and standing for culture, wherein cells grow out of the tissue block after about 3 weeks;

(3) cell purification: inoculating for about 7 weeks, transferring to generation 1, digesting with 0.25% trypsin, inoculating the cell suspension into a culture dish, observing under an inverted phase contrast microscope to see that part of cells adhere to the wall, and pouring the cell suspension into another culture bottle for continuous culture; repeating the passage for 3 times according to the method to completely separate the tumor cells from the fibroblasts;

(4) subculturing: when the cells are full of the bottom of the bottle, sucking out the culture solution, rinsing with PBS buffer solution, digesting with 0.25% trypsin, and carrying out passage for 30 generations according to the ratio of 1: 3;

(5) biological characteristic identification is carried out to obtain the human placenta trophoblastic tumor cell line PSTT-1 with the preservation number of No. 10882.

4. The human placental site trophoblastic tumor cell line according to claim 3, wherein said preparation method uses in vitro culture conditions: RPMI1640 cell culture medium containing 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% HEPES and 1% double antibody, the culture temperature is 37 ℃, and the gas environment is 5% CO₂And/95% air, and the humidity is saturated humidity.

5. Use of the human placental site trophoblastic tumor cell line of claim 1 for the preparation of models for the study of the etiology, metastatic mechanisms, resistance mechanisms and clinical intervention of placental site trophoblastic tumor diseases.