CN1935990A - Human placental nutritive layer cell line and its use - Google Patents

Abstract

The invention relates to human placenta trophoblastic cell series which is conserved in China Committee for Culture Collection of Microorganisms on September 22th in 2005 with CGMCC No.1461 conserving number. It includes the following steps: separating the trophoblastic cell form the placenta tissue which is pregnant for 6-7 weeks; filtering the cells with multiplication capacity; processing subunit gene transfection by telomere enzyme to gain four long life cell strains. The cell series has normal multiplication capacity and biological chemical function, active cell growth, and reaches the 210 generation. Thus it can be used as target cell model for filtering antifertility drug and curing pregnant correlative disease medicine, provide ideal vitro model for further studying trophoblastic cell, lay important foundation for studying embryo implantation and pregnant physiology and pathological mechanisms.

Description

A kind of human placental nutritive layer cell line and uses thereof

Technical field

The present invention relates to a kind of clone, specifically relate to a kind of people's placenta cells trophocyte system, and uses thereof.

Background technology

In the gestation process, placenta is the important endocrine organ who maintains nutrition and exchange of substance between parent and the fetus, is the important leverage that fetation and pregnancy maintenance are able to success.Topmost composition cell is the trophocyte in the placenta, comprise that having of monokaryon enliven the plamoditrophoblast cell (STB) that the cell trophoblastic cell of multiplication capacity (CTB) and multinuclear are born main endocrine function, STB is merged and forms through cytolemma by CTB.

Initially to end, most of function of placenta is carried out by the trophocyte from gestation.When the embryo implants, rely on trophocyte and endometrial identification, adhere to and invade profit, plant and anchor to intrauterine; The while trophocyte secretes multiple hormone, cytokine etc. and keeps embryo's normal development and nutrition and the exchange of substance between female tire; When gestation finished, the trophocyte impelled placenta to peel off from the uterus by producing proteolytic enzyme etc.Clinically, many pregnant relative diseases are all relevant with trophocyte's dysfunction, as spontaneous abortion, preeclampsia, hydatidiform mole, chorioepithelioma etc.Therefore, in the embryo implants and gestation takes place physiology and pathological study, the research that trophocyte's function is regulated is crucial key link.

Since the nearly century, a lot of scholars are devoted to set up the vitro culture system of people's placental trophoblasts, can simulate the research model of the function adjusting of trophocyte under the normal physiological conditions with acquisition.The most frequently used is the chorion epithelioma cancerous cell line, as Jar, JEG, BeWo, NUC-1, HCCM-5 etc. are at document 1:Ringler GE, Strauss JF III (1990) In vitro system for the study of human placental endocrine function.Endocr Rev 11, described in the 105-123, though they have certain similarity with normal trophocyte on some endocrine function, most of function regulate especially breed the adjusting aspect and normal cell completely different.Ho etc. are at document 2:Ho CK, Chiang H, Li SY, Yuan CC, Ng HT (1987) Establishment and characterization of atumorigenic trophoblast-like cell line from a human placenta.Cancer Res 47,3220-3224. middlely reported a kind of trophoderm like cell system-TL that comes from normal mature placenta, but this cell can not synthesize the hormone in multiple placenta source, and cellular form and characteristic heterogeneity, has the aneuploid karyotype, in the nude mice of immunodeficiency, can cause tumour and produce, thereby be considered to the improper trophocyte system of a group tumourization.1992, Lei etc. are at document 3:Lei KJ, Gluzman Y, Pan CJ, Chou JY (1992) Immortalization ofvirus-free human placental cells that express tissue-specific functions.Mol Endocrinol 6, among the 703-712, monkey disease poison SV40 large T antigen gene transformation people cell trophoblastic cell with responsive to temperature type, obtain a few strain cells, they are under 37 ℃ culture condition, can be with the uncontrolled propagation of the mode of tumour cell, under 40 ℃ of culture condition, the propagation mode is similar to normal cell, but its hormone secretion and regulative mode be not quite similar with normal trophocyte, so these cells still can't be as normal cell trophocyte's representative.In fact, up to the present, also do not set up the normal trophocyte of people system in the world, although numerous scholars has paid great effort.This mainly be since with the people as research object, ethics problem causes obtaining this research material of human placenta certain obstacle (especially in western countries), simultaneously because people's individual difference is very big, the result who has caused coming from different investigators is conflicting.

Summary of the invention

The object of the present invention is to provide human placental nutritive layer cell line a kind of immortalization, that keep normal trophocyte's function, and uses thereof.

The objective of the invention is to realize by the following technical solutions:

Human placental nutritive layer cell line provided by the invention is set up by following step: be separated to cell trophoblastic cell from people's normal pregnancy placenta tissue in 6-7 week; Therefrom filter out cell mass with multiplication capacity; Through the telomerase catalytic subunit gene transfection, 4 long-life cell strain B6Tert-1, B6Tert-2, B6Tert-3 and B6Tert-4 have been obtained with telomerase activation.

The B6Tert-1 cell that the present invention sets up is preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on September 22nd, 2005, and its preserving number is CGMCC No.1461.

The B6Tert-1 cell is carried out the evaluation of biochemical and multiplication characteristic

Cellular form is observed: inverted phase contrast microscope is observed down, and the B6Tert-1 cell keeps monolayer growth in culture dish, be the epithelioid cell of multiangular, and cell is a monokaryon, contains 1-5 kernel in each cell; In the long-term cultivation process, seldom observe syncyte; When cell inoculation density was low, some cells can be stretched and draw very longly, with the cell of the pseudopodium spline structure contact apart from each other that extends, as shown in Figure 1.The cellular form of this homogeneous remains to 210 from generation to generation always, and can continue continuity.

The B6Tert-1 cell that the present invention sets up possesses Normocellular multiplication characteristic and human cell trophocyte's biochemical function, and in nude mice tumorigenesis not; While cell active growth, reached 210 from generation to generation, it is a kind of human normal cell line trophocyte system of immortalization, the target cell model that can be used as screening antifertility drug and the pregnant relative disease medicine of treatment, also provide the ideal external model, be the important foundation of research embryo implantation and gestation generation physiology and pathomechanism for further studying human trophocyte cell's function.

Description of drawings

Fig. 1 is for to differ observed B6Tert-1 cell under the inverted microscope; Wherein, (A) be 80 from generation to generation B6Tert-1 cells; (B) be the B6Tert-1 cell of inoculum density when low, the pseudopodium spline structure (*) of extended length; Scale is 25 μ m.

Fig. 2 is that the nucleic acid blot analysis shows the expression of hCG α and hCG β mRNA in the B6Tert-1 cell, and as positive control, house-keeping gene GAPDH is as internal reference with the RNA in placental villi (Placenta villi) source.

Fig. 3 is that RT-PCR analyzes the expression pattern that shows multiple MMPs and TIMPs mRNA in the B6Tert-1 cell, and as positive control, house-keeping gene GAPDH is as the internal reference of PCR reaction with the RNA in placental villus tissues (Placenta vill) source.

Embodiment

The foundation of embodiment 1.B6Tert-1 clone

Isolated cell trophocyte from the pregnant 6-7 artificial abortion placenta in age in week (also can be referred to as fine hair this moment) at first.Obtain aseptic chorionic villi 3-5 from the birth control outpatient service, in ice bath, take back the laboratory; Below operation is all carried out on the aseptic technique platform.Tissue is placed the PBS damping fluid that is cooled to 4 ℃ in advance, the clot that flush away is infected with; Tissue is transferred in the fresh PBS damping fluid, separated fine hair and basilar membrane down and discard basilar membrane in anatomical lens; Collect fine hair and shred into 2-3mm ³Fritter, transfer in the 50ml centrifuge tube, add the trypsin U.S. Sigma company contain 0.2-0.3%) PBS damping fluid 20ml, behind the mixing in 4-9 ℃ of digestion 30-50min; It is centrifugal that (1000rpm discards after 5min) and asks liquid, adds the DNA enzyme (U.S. GIBCO company) that fresh FD nutrient solution of 20ml and final concentration are 20-40IU/mL, and normal temperature is digestion 5-15min down; Centrifugal (1000rpm discards after 5min) and asks liquid, adds the fresh FD nutrient solution suspended tissue of 20-40ml, through 150 order nylon net filters, and centrifugal (1000rpm, 5min) collecting cell; Add 2mlFD nutrient solution suspension cell, (lower floor is the 2.5%BSA solution that 2ml is formulated in the FD nutrient solution to the BSA density gradient of while preparation 1.25%-2.5% in the 10ml centrifuge tube, the upper strata is the 1.25%BSA solution that 2ml is formulated in the FD nutrient solution), cell suspension is carefully added BSA density gradient upper strata, be kept upright and sedimentation 30-60min at room temperature; Collect undermost cell, after suspending with the FD nutrient solution, according to 2-4X10 ⁵The density of/ware is inoculated in and scribbles 6-10 μ g/cm ²Cellmatrix (type i collagen, Japan Osaka biochemical research institute) in the  35mm culture dish, used conditioned medium is for having added the FD nutrient solution of 10ng/mL Urogastron (U.S. Sigma company), 1 μ g/mL Regular Insulin (U.S. Sigma company), 2mmol/L glutamine and 0.1%BSA (U.S. Sigma company), 1.5-2ml/ ware, in 37 ℃, 5%CO ₂Cultivate in the incubator of condition.Behind the 24h, change fresh conditioned medium.

From cultured cells, screen the cell mass that can keep multiplication capacity then.Under above-mentioned culture condition, cultured continuously cell 1-2 month, nutrient solution of replacing in every 24-48 hour.There is a large amount of cells floating and dead during this time, but has small amounts of cells to keep adherent growth, and the slow propagation of beginning.When treating that the 70-90% of confluent culture ware bottom surface is arrived in these cell proliferations, carry out had digestive transfer culture, concrete grammar is: discard nutrient solution, wash culture dish once with the PBS damping fluid, add 1ml and contain the tryptic PBS liquid of 0.05-0.1%, culture dish is placed 5-10min on ice, discard trypsin solution, add 100-200 μ l PBS liquid, culture dish is placed 5-10min in 37 ℃ of incubators, taking-up also adds an amount of conditioned medium suspension cell, is inoculated in other 2-3 the new  35mm culture dish that scribbles Cellmatrix, continues to cultivate according to the method described above.

In cell, import telomerase catalytic subunit gene (htert) subsequently.Cultivate above-mentioned proliferative cell to the 10-20 generation, when treating the 40-60% of cell confluent culture ware bottom surface, carry out gene transfection.Concrete grammar is:

1) prepares at least 2 ware cells;

2) in a tubule, 6 μ l FuGene6 (German Boehringer Mannheim company) reagent and 94 μ lFD nutrient solutions is evenly mixed, under room temperature, place 5min;

3) get two tubules in addition, adding is dissolved in the 10-40 μ g htert expression plasmid pGRN145 (U.S. Geron company) in the 5-20 μ l water, and another adds isopyknic water;

4) with 2) in the FuGene6 reagent of dilution dropwise add 3 respectively) in two tubules in, simultaneously softly shake tubule with mixing; Room temperature is placed 15min;

5) with 4) in the reagent of mixing add 1 respectively) in ready two culture dish, note guaranteeing evenly to drip at each position of culture dish; The cell that wherein adds FuGene 6 reagent and htert expression plasmid pGRN145 mixture is an experimental group, and another adds the negative control group of cell of FuGene 6 reagent and water mixture;

6) cell being put into incubator cultivated 5-10 hour;

7) changing fresh conditioned medium continues to cultivate 24 hours;

8) add 3-10 μ g/ml Hygromycin (U.S. SIGMA company), cultured continuously 5-10 days, and by changing the cell that nutrient solution discards death.

Carry out the clone and the amplification work of cell afterwards.After cell process said gene transfection and drug screening were handled, the cell of control group was almost all dead, and experimental group has the cell survival of 1-5%, and presented little clonal growth.By microscopic examination, mark these little clones, after discarding nutrient solution, the bottom is scribbled clone's ring cowling of thin layer vaseline oil on the clone of mark, according to foregoing method each clone's intra-annular cell dissociation is got off, be inoculated in respectively in 24 well culture plates that scribble Cellmatrix, add the 1ml conditioned medium and carry out the routine cultivation.Treat cell grow into be paved with 70-90% culture plate bottom surface after, digest once more and be inoculated in the  35mm culture dish that scribbles Cellmatrix, behind the cell confluent culture ware, carry out had digestive transfer culture according to 1: 3 ratio, cell is increased gradually.

In the long-term cultivation process, in order to preserve cell, it is freezing to need pair cell to carry out.Behind the cell dissociation, move in the 10ml centrifuge tube, and interpolation 5-10ml FD nutrient solution, it is centrifugal that (1000rpm 5min) and abandoning supernatant, is cooled to 4 ℃ cell freezing liquid (adding 10% foetal calf serum and 10% dimethyl sulfoxide (DMSO) in the FD nutrient solution) suspension cell in advance with 1ml, move in the cell cryopreservation pipe, place 4 ℃ of 30min successively, after-20 ℃ of 60min spend the night with-80 ℃, change in the liquid nitrogen and preserve.

Through said process, obtain 4 stable clonal cell lines altogether, be named as B6Tert-1, B6Tert-2, B6Tert-3 and B6Tert-4 respectively.Through nearly 3 years cultivation, the B6Tert-1 cell reached for 210 generations (cell fission once is a generation) at present, any old and feeble feature do not occur.The generally accepted viewpoint of cell biological educational circles is that cell survival surpasses 100 and is from generation to generation and reached immortalization, so the B6Tert-1 cell that we set up has been the cell of immortalization.This B6Tert-1 cell is preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on September 22nd, 2005, and its preserving number is CGMCC No.1461.

Embodiment 2. cell proliferation specificity analysises

1) rate of propagation of cell

B6Tert-1 (80PDs) cell is according to 10 ⁴Individual/ware is inoculated in the  35mm culture dish that scribbles Cellmatrix, all gets three wares at random from 1-11 days every days and carries out cell counting, and draw the cell proliferation curve thus.The result shows that the cell doubling time of B6Tert-1 is 36 hours; Cell reached converging state (be that cell closely contacts, do not have unnecessary space in the culture dish) in the 9th day, cell proliferation rate slows down and finally stops propagation at this moment.This phenomenon is become " contact inhibition " of cell growth, is one of Normocellular multiplication characteristic.

2) adjusting of Urogastron (EGF) and transforming growth factor (TGF) β on cell proliferation:

With the B6Tert-1 cell according to 5 * 10 ⁵Individual/ware is inoculated in the  35mm culture dish that scribbles Cellmatrix, and after 48 hours, each stimulating dose is chosen three ware cells respectively, carries out cell counting with 1-100ng/ml EGF irritation cell.The result shows that B6Tert-1 cell proliferation is regulated by EGF, and 1-100ng/ml EGF can stimulate B6Tert-1 cell proliferation, and along with the increase of EGF concentration, cell proliferation rate is also accelerated, and the maximal stimulation effect of EGF on cell proliferation is 100ng/ml.

With the B6Tert-1 cell according to 5 * 10 ⁵Individual/ware is inoculated in the  35mm culture dish that scribbles Cellmatrix, adds 10ng/ml EGF, adds 0,0.1,1 and 10ng/ml TGF β simultaneously, handles after 48 hours, and each stimulating dose is chosen three ware cells respectively, carries out cell counting.The result shows that in the B6Tert-1 cell, itself does not influence cell proliferation TGF β, but the partly effect of antagonism EGF; 10ng/ml TGF β effect made the cell proliferation amount that EGF causes in two kinds of cells all reduce about 35% in 48 hours.

As seen, the propagation of B6Tert-1 cell is subjected to the forward of different somatomedins and secondary to regulating effect respectively, has stronger controllability.

Embodiment 3. cell tumorigenicity are analyzed (Tumorigenicity)

To grow to 80 generations and 210 B6Tert-1 cell from generation to generation according to 10 ⁷Cell/100 μ l distinguish subcutaneous injection to nude mice, each 3 nude mice of injection cell from generation to generation.Observe the tumor growth situation in the nude mice.After three months, not seeing has any tumour to form in the nude mice.As seen, the B6Tert-1 cell does not possess tumorigenicity.

Embodiment 4. chromosome analysises

80 generations and 210 B6Tert-1 cell is from generation to generation carried out chromosome analysis, by analyzing the cell chromosome of about separately 100 metaphases, find that they all have normal human cell's amphiploid caryogram, contain 46 karyomit(e)s, comprising sex chromosome X and Y.As seen, the karyomit(e) of B6Tert-1 cell is the normal cell caryogram.

Embodiment 5. trophocyte's specificity analysises

Select for use 80-100 B6Tert-1 cell from generation to generation to carry out the biochemical characteristic analysis, the result is described below respectively:

1) cytokeratin (Cytokeratin, expression CK)

Cytokeratin is a cytoskeletal protein special in the cell of epithelial origin, can be divided into multiple hypotype.People's placental trophoblasts is the cell of epithelial origin, has CK7, Ck8, various kinds of cell Keratin sulfate such as CK18.

Experiment adopts immunocytochemistry to identify the proteic distribution of CK8 in the B6Tert-1 cell.The B6Tert-1 cell cultivated is fixed 20 minutes with 0.125% glutaraldehyde (containing 3% sucrose) in 37 ℃, hatch H then in 0.3% ₂O ₂In to remove endogenic peroxidase.Dripped mouse-anti people CK8 antibody (1: 100; SIGMA) back is in 4 ℃ of overnight incubation.Second day, wash cell 3 times with PBS after, add the Polymer (DAKOEnvision of horseradish peroxidase-labeled ^TMTest kit provides) incubated at room 30min, PBS washes 3 times.Add diaminobenzidine colour developing 5-15min, the tap water flushing is reacted with color development stopping.Carry out the observation of positive signal at microscopically.Found that the immune positive staining that all presents CK8 above 99% B6Tert-1 cell, show that they all are the epithelial cell sources, meet normal trophocyte's characteristic.

2) expression of human chorionic gonadotrophin (hCG)

Human chorionic gonadotrophin hCG is the hormone of the special generation of people's placental trophoblasts, and this albumen mainly is made up of two subunits, i.e. hCG α and hCG β.Under the normal circumstances, cell trophoblastic cell is merely able to synthetic hCG α, does not possess hormone function, has only the plamoditrophoblast cell can synthesize two kinds of subunits, the hCG albumen of composition function.Therefore, the expression of hCG α can be used as the sign of cell trophoblastic cell, and the expression of hCG β is one of sign of cell trophoblastic cell generation zoariumization.The means that detect these developed by molecule are nucleic acid blot analyses.Its concrete operation is as follows:

I. the interior or interior extraction of RNA always of tissue of cell

Press TRIzol reagent (Invitrogen, the U.S.) the total RNA that extracts in cell and the tissue is described: by 10 ⁶The ratio of cell/ml TRIzol reagent adds TRIzol reagent in cell; For tissue, then add TRIzol reagent according to the ratio of 100mg tissue/mlTRIzol reagent, place on ice, carry out homogenate, with the liquid collecting after the homogenate in centrifuge tube; Above-mentioned sample adds 200 μ l chloroforms after room temperature is placed 5min, use forced oscillation 25 seconds, and room temperature is placed 3min; 4 ℃ centrifugal 13, and 000rpm/15min makes the liquid layering; The water that the upper strata is contained RNA is drawn in the new centrifuge tube, adds the 0.5ml Virahol, and rolling is even gently, and room temperature is placed 10min and made the RNA precipitation; 4 ℃ centrifugal 13, and 000rpm/10min makes RNA be pressed into fritter in the centrifuge tube prodelta clay; The Virahol that inclines, with 70% washing with alcohol RNA precipitation, 4 ℃ are centrifugal 10, and 000rpm/5min collects RNA; Drying at room temperature adds sterilized water dissolving RNA.

Ii. RNA gel electrophoresis and transfer

The 1.6g agarose is sneaked in the 100ml deionized water, heating for dissolving, to be cooled after about 60 ℃, add 32ml 5 * MOPS damping fluid and 29.2ml formaldehyde, mixing is poured in the gel supporting plate, solidifies under the room temperature 1 hour, makes 1% running gel.With the total RNA sample of 25 μ g (9 μ l), 4 μ l, 5 * MOPS, 7 μ l formaldehyde, 20 μ l deionized formamides and 4 μ l, 10 * sample loading buffer (50% glycerine, 1mM EDTA (pH8.0), 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene cyanogen) mix, 65 ℃ of heat denatured 10min place cooled on ice 5min rapidly.Sample is added the point sample hole of gel,, treat that tetrabromophenol sulfonphthalein stops electrophoresis (about 2-3h) when migrating to the gel bottom according to 4V/cm constant voltage electrophoresis.Use the rinsed with deionized water gel under the room temperature, 10min * 2 time; With 20 * SSC damping fluid rinsing gel, room temperature, 15min * 2 time.Clip is the Hybond of size suitably ⁺Nylon membrane (U.S. Pharmacia company) soaks 5min in 20 * SSC; Glue is superimposed upon on the film, places vacuum transhipment instrument (Pharmacia), 50mBar shifts 1-3h.Nylon membrane after the transfer in 2 * SSC rinsing once, crosslinked 10sec in UV-crosslinked instrument makes RNA and film covalent attachment.Nylon membrane can place 4 ℃ of preservations.

Iii. probe mark and purifying

According to the cDNA sequence of hCG α, hCG β gene and house-keeping gene GAPDH among the Genebank, it is as follows to design primer sequence respectively, and is synthesized by the Shanghai company of widely collecting deeply:

HCG α: upstream primer 5 '-CGAAGGGTTAGTGTCGAGC-3 '

Downstream primer 5 '-ATGGACTTGGAAGGCTGG-3 '

HCG β: upstream primer 5 '-TTCCTACACCCTACTCCCTGT-3 '

Downstream primer 5 '-CCGGCAGGACCCCCTGCAGCA-3 '

GAPDH: upstream primer 5 '-CTCAGACACCATGGGGAAGGTGA-3 '

Downstream primer 5 '-ATGATCTTGAGGCTGTTGTCATA-3 '

In the following reaction system of mixing (cumulative volume 20 μ l) on ice, carry out reverse transcription reaction:

The total RNA of human placenta (1 μ g/ μ l) 2 μ l

Oligomerization dGMP (500 μ g/ml, Promega company) 1 μ l

Thymus nucleic acid (10mmol/L, Invitrogen) 1 μ l

Aseptic deionized water (Invitrogen) 12 μ l

Behind 65 ℃ of heat denatured 5min, place 2min on ice rapidly; Add 4 μ l, 5 * reverse transcription damping fluids (Invitrogen) again, mixing is hatched 2min for 42 ℃; Add 1 μ l Superscript II reversed transcriptive enzyme (Invitrogen) again, mixing is hatched 50min for 42 ℃.After 70 ℃ of heating 15min deactivation reactions, reaction product (being the cDNA template) is kept at-20 ℃.

Subsequently in the following PCR reaction system of mixing (cumulative volume 50 μ l) on ice:

10 * PCR damping fluid (Promega), 5 μ l

MgCl ₂(25mmol/L，Promega) 2μl

DNTP (each 10mmol/L) 1 μ l

Upstream primer (10 μ mol/L) 1 μ l

Downstream primer (10 μ mol/L) 1 μ l

Deionized water 37 μ l

The cDNA template 1 μ l that reverse transcription reaction obtains

Taq DNA Polymerase (Promega) 2μl

Behind the mixing, on the PCR instrument, carry out following amplification program: 94 ℃/3min → [94 ℃/30 sec → 56-67 ℃ (and because of different amplification genes different)/30sec → 72 ℃/50sec] * → 72 ℃/5min of 30 circulations.

The PCR product is delivered Shanghai and can the company of widely collecting be checked order deeply, and sequence meets the probe that can be used as nucleic acid blot hybridization of hCG α and hCG β gene cDNA fragment.

Adopt prime-a-gene test kit (Promega) according to random priming carry out probe [α- ³²P] radio-labeling.Mixed following composition:

5 * mark damping fluid (Promega), 10 μ l

DNTP mixture (Promega) the 2 μ l of no dCTP

Be heated to the PCR product 25ng of 98 ℃ of sex change

The BSA of nuclease free (Promega) 2 μ l

[α- ³²P] dCTP (3000Ci/mmol, the inferior brightness in Beijing company) 5 μ l

Archaeal dna polymerase Klenow fragment (Promega) 5U

The water that adds nuclease free is to final volume 50 μ l

Mixing, room temperature reaction 1h, can obtain [α- ³²P] the cDNA probe of mark.

Iv. hybridization

Nylon membrane is adherent to be placed in the hybridization Glass tubing, adds the 10ml prehybridization solution, hatches 4h for 65 ℃; Discard prehybridization solution, add fresh hybridization solution of 5ml (adding 10% T 500 in the prehybridization solution) and sex change probe (probe that mark is good heats 10min in 95-100 ℃ of water-bath, place on ice rapidly), mixing, 65 ℃ of hybridization spend the night (15-20 h).Hybridization solution is poured in the safe waste liquid cylinder, and (1 * SSC, 0.1%SDS) room temperature washing Hybond membrane 30min is with the film washing liquid II (NaH of 25mM with film washing liquid I ₂PO ₄.2H ₂O, the Na of 25mM ₂HPO ₄.12H ₂O, the EDTA of 1mM 1%SDS) washes film 30min * 3 time in 65 ℃.Nylon membrane after the hybridization is encapsulated in the preservative film, is stacked on the X-ray sheet (Japanese fuji) in the darkroom, places the compressing tablet box, and-80 ℃ were exposed 1～7 day.

V. develop a film

In the darkroom X-ray sheet is developed in developing solution, safety light (development red light) is observed the development degree down, in time ends to develop, and places stop bath photographic fixing 5min then, and tap water washes down, and dries.

By above-mentioned nucleic acid blot crossover process, finding has the hCG alpha expression of certain level in the B6Tert-1 cell, does not express and detect hCG β, as shown in Figure 2.Show that the B6Tert-1 cell is a cell trophoblastic cell, zoariumization does not take place.

3) distribution of gonadotropin releasing hormone (GnRH)

The normal cell trophocyte can produce a certain amount of GnRH.At first prepare the anti-people GnRH of rabbit polyclonal antibody according to ordinary method.With GnRH decapeptide and BSA coupling connection, behind the abundant mixing of Fu Shi Freund's complete adjuvant, according to the subcutaneous multi-point injection new zealand rabbit of the protein content of 150 a μ g/ rabbit, the two all booster shots of every interval once, booster shots are 4 times altogether, and from a week beginning to measure serum antibody titer after the immunity for the third time, rabbit is put to death in last immunity two all backs, and the heart blood sampling, separation of serum is frozen in-20 ℃.

By being similar to the immunocytochemical assay method that cytokeratin detects, analyze the distribution situation of GnRH in cell, the result shows that the B6Tert-1 cell above 98% presents GnRH immunity positive reaction, signal mainly is positioned in the tenuigenin.This further specifies the characteristic that the B6Tert-1 cell possesses cell trophoblastic cell.

4) matrix metalloproteinase (matrix metalloproteinases, MMPs) and tissue inhibiting (tissueinhibitor of MMPs, expression TIMPs)

MMPs and TIMPs are proteolytic enzyme and the inhibitor that cell trophoblastic cell produces, and their generation makes the trophocyte possess certain profit ability of invading.This research is used reverse transcription-polymerase chain reaction (RT-PCR) and is detected the ability that the B6Tert-1 cell produces MMPs and TIMPs.

According to the GenBank accession number of goal gene, retrieve its cDNA sequence.Utilize Omiga software to carry out design of primers (seeing the following form 1), can betting office synthesize by the Shen, Shanghai.

Table 1 is used for the primer sequence and the reaction conditions of PCR reaction

The gene title	Primer sequence (F: upstream primer; R: downstream primer)	Annealing temperature (℃)	Cycle number
				MMP-2	F：5’-ACCTGGATGCCGTCGTGGAC-3’ R：5’-TGTGGCAGCACCAGGGCAGC-3’	56	30
MMP-9	F：5’-GTGCTGGGCTGCTGCTTTGCT-3’ R：5’-GTCGCCCTCAAAGGTTTGGAA-3’	67	30
				MMP-14	F：5’-ATTGAATTCAGCCCCGAAGCCTGGC-3’ R：5’-ATTGAATTCTCAGGCCCTCACGGA-3’	57	28
TIMP-1	F：5’-GCGTTATGAGATCAAGATGACC-3’ R：5’-AGGCTTCAGCTTCCACTCC-3’	58	28
				TIMP-2	F：5’-ATCACCCTCTGTGACTTCATCG-3’ R：5’-ATTCATGCTGTTTCCAGGAAGG-3’	58	28
TIMP-3	F：5’-TGCCAGAAAGAATGAGGAACC-3’ R：5’-AGAGAGGGTGCTGACGGTGTT-3’	56	30

Carry out reverse transcription reaction and PCR reaction subsequently, concrete grammar as mentioned above.

After the PCR reaction finishes, the 0.2g agarose is added in the 20ml TE damping fluid, after the heating for dissolving, pour in the gel supporting plate, make 1% sepharose; 10 μ l PCR reaction product are added in the well of gel,, treat that tetrabromophenol sulfonphthalein stops electrophoresis (about 1-2h) when migrating to the gel bottom according to 4V/cm constant voltage electrophoresis.Under ultraviolet lamp, detect the electrophoresis result of PCR product, and with the imaging of gel UV scanning analyser (U.S. UnitedBio company), as shown in Figure 3, the expression pattern of multiple MMPs and TIMPs mRNA in the RT-PCR analysis demonstration B6Tert-1 cell, the RNA in placental villus tissues (Placenta vill) source is as positive control, and house-keeping gene GAPDH is as the internal reference of PCR reaction.

By above-mentioned RT-PCR reaction, confirm that MMP-2 ,-9 is arranged in the B6Tert-1 cell ,-14 and TIMP-1 ,-2 ,-3, the expression of-4mRNA possesses certain profit ability of invading.

The evaluation of above-mentioned cell proliferation and biochemical characteristic shows that the B6Tert-1 cell possesses Normocellular multiplication characteristic and human cell trophocyte's biochemical function, and in nude mice tumorigenesis not; The cell active growth has reached 210 from generation to generation simultaneously.Therefore, the B6Tert-1 cell is the human normal cell line trophocyte system of immortalization.

The prescription of the several solns of mentioning among the present invention is as follows:

1.FD nutrient solution: after following medicine dissolving, be stored in 4 ℃ through 0.22 μ m membrane filtration is also aseptic.

F12 (GIBCO company) 10.6g

DMEM(GIBCO) 13.4g

Penicillin 200,000 units

Streptomycin sulphate 200,000 units

HEPES 1.686g

NaHCO ₃ 2.4g

Deionized water 2000ml

2.PBS damping fluid: after following medicine dissolving, be stored in 4 ℃ through 0.22 μ m membrane filtration is also aseptic.

NaCl 8g

KCl 0.3g

Na2HPO4 1.83g

KH2PO4 0.02g

Glucose 2g

Deionized water 1000ml

3.5XMOPs damping fluid:

Sodium acetate 2.72g

MOPs 10.3g

0.5M EDTA 5ml

Above-mentioned medicine is woven in the 493ml deionized water, add 2ml 10NNaOH mixing after, through 0.22 μ m membrane filtration and keep in Dark Place in normal temperature.

4.20XSSC damping fluid: following medicine is dissolved in the 1000ml deionized water, and to regulate pH value with 1N NaOH be 7.0.Normal temperature is preserved.

NaCl 175.3g

Trisodium citrate 88.2g

5. prehybridization solution: following agent dissolves in deionized water, and is settled to 100ml.Be stored in 4 ℃.

SDS (sodium laurylsulfonate) 7g

Na2HPO4-12H2O 4.9g

NaH2PO4 0.988g

0.5M EDTA 0.2ml

BSA 1g

Methane amide 15ml

6.TE damping fluid:

0.5M Tris damping fluid (pH8.0) 2ml

0.5M EDTA 0.2ml

Deionized water 98ml

7.0.5M Tris damping fluid (pH8.0)

Dissolving 60.55g Tris adds about 21ml concentrated hydrochloric acid and regulates pH to 8.0 in the 800ml deionized water, is settled to 1000ml with deionized water.

Claims (3)

1, a kind of human placental nutritive layer cell line is preserved in " China Committee for Culture Collection of Microorganisms common micro-organisms center " on September 22nd, 2005, and its preserving number is CGMCC No1461.

2, human placental nutritive layer cell line as claimed in claim 1, it is to set up by following step: be separated to cell trophoblastic cell from people's normal pregnancy placenta tissue in 6-7 week; Therefrom filter out cell mass with multiplication capacity; Through the telomerase catalytic subunit gene transfection, 4 long-life cell strains have been obtained with telomerase activation.

3, the described human placental nutritive layer cell line of a kind of claim 1 is as human trophocyte cell's functional study with screen antifertility drug and treat application in the target cell model of pregnant relative disease medicine.

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