CN102146358A - Building method for luteal cell system of goat - Google Patents
Building method for luteal cell system of goat Download PDFInfo
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- CN102146358A CN102146358A CN 201110008726 CN201110008726A CN102146358A CN 102146358 A CN102146358 A CN 102146358A CN 201110008726 CN201110008726 CN 201110008726 CN 201110008726 A CN201110008726 A CN 201110008726A CN 102146358 A CN102146358 A CN 102146358A
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Abstract
The invention belongs to the field of a biological and animal cell system and in particular relates to a building method for a luteal cell system of a saanen dairy goat in the early gestation period. In the method, luteal tissues of the healthy saanen dairy goat which is pregnant for 6-8 weeks are used as a cell source, the luteal tissues are digested by collagenase to obtain discrete luteal cells, and the luteal cells are expansively cultured and purified in vitro. Human telomerase reverse transcriptase gene is transfected and purified by liposome in the purified luteal cells and is screened by resistance gene, and monoclone is selected for expansion culture. Then the identification is carried out through histochemical or cytochemical staining. The cells of the luteal cell system are polygonal or shuttle-shaped, and 3beta-hydroxysteroid dehydrogenase staining is positive; in addition, essential properties of the original luteal cells are maintained.
Description
Technical field
The invention belongs to biological field of animal cell lines, be specifically related to the establishment method that a kind of goat lutein cell is.
Background technology
Corpus luteum is the temporary endocrine tissue that Mammals ovulation back forms in ovary, keep normal reproductive function for Mammals and have vital role, it is controlled by the adjusting of pituitary gland gonad-stimulating hormone in addition, and its excretory steroid hormone mainly is a progesterone.It mainly acts on: (1) stimulates the secretion of uterine gland, and metremia swelling and Uterine mucosa plumpness guarantee the zygote implantation; (2) guarantee the gestation survival of fetus in early days.In addition to hypothalamus-pituitary gland axle, ovary, organs such as uterine tube and mammary gland have regulating effect.Influence the secretion of lutein cell progesterone if Recent study is found corpus luteum graviditatis dysfunction or other drug, then can influence the survival of maintenance of pregnancy and fetus.
Corpus luteum is as one of tissue that has periodically growth and degradation characteristics in the jenny body of growing up, and the regularity variation of its weave construction is regulated and excretory mechanism is the emphasis of many researchs always, also is one of the hottest endocrine organ of Recent study.Lutein cell in form, all has very big difference in the vitro culture limited time between the different algebraically cells on biological nature and the gene expression dose in addition, and this has also caused the experimental result between the different laboratory to be difficult to compare.
Therefore the lutein cell system that sets up a kind of biological property of energy maintenance lutein cell of former generation and the immortalization that long-term stability goes down to posterity is significant for the physiological regulation function of illustrating lutein cell.
Because the probability of spontaneous immortalization is very little, therefore sets up clone method commonly used at present and mainly contain: radioactivity, the transfection of oncogene and proto-oncogene, DNA oncogenic virus.These methods usually make the cell caryogram instability of immortalization, and phenotypic alternation and have many shortcomings such as tumorigenicity has limited its widespread use.
Summary of the invention
The purpose of this invention is to provide the goat lutein cell system of biological properties such as a kind of energy maintenance lutein cell progesterone secretion of former generation, is the growth of research lutein cell, and the molecular mechanism that Physiology and biochemistry and progesterone secretion are regulated has improved cell model and research tool.
Another object of the present invention provides the method for separation and purifying goat lutein cell of former generation.
The present invention sets up goat lutein cell system by following method and step
1 former being commissioned to train supported and purifying
By checking the fetus size and observing development degree and judge the Gestation period, the collagenase digesting luteal tissue makes it become individual cells or cell mass, and the inoculating cell bottle cultivates, and citric acid-trysinization, and inverted microscope is observed down, when about 20%～40% cellular contraction is rounded, add nutrient solution and stop digestion, and blow and beat, remove the cell of suspension, continue to cultivate residue, and repeat several times.Reach more than 95% until purity.
Identify au bleu around the nucleus of positive cell by the histocytochemistry pair cell.(should be blue like this)
2 transfections and screening
With liposome 2000 human telomerase reverse transcription gene is imported in the lutein cell, adds (450 μ g/ml) G418 after the transfection and screen 2-3 week, wait mono-clonal to form after, the picking mono-clonal is also identified with the histocytochemistry method.
Positive cell is carried out enlarged culturing, went down to posterity once in 3 days, the rate that goes down to posterity 1: 2 treats to carry out when cell passed to for 30 generations qualification test, and measures the growth curve of transfection front and back goat lutein cell.
The table that RT-PCR detects lutein cell human telomerase reverse transcriptase gene mRNA arrives, and the ELISA method is checked telomerase activation.
The expression of key gene in the lutein cell after the RT-PCR detection transfection is with the amount of lutein cell secretion progesterone after the transfection of radioimmunology (RIA) mensuration.
The variation of lutein cell karyomit(e) quantity after the karyotyping transfection.
Whether lutein cell had the feature that tumour transforms after soft agar test and tumor after being inoculated into nude mice test detected transfection; As suspension growth and tumorigenicity.
The goat lutein cell system that utilizes aforesaid method to set up can have following biological nature external long term growth and stable going down to posterity:
(1) with the primary cell plesiomorphism, the cell of immortalization is multiangular or shuttle type;
(2) the exogenous human telomerase reverse transcriptase gene can be in the lutein cell of transfection stably express, and high telomerase activity is arranged, the multiplication capacity of cell obviously increases;
(3) this clone has kept some key characters of former generation lutein cell, as the secretion progesterone, expresses the key gene in the lutein cell: StAR, P450scc, 3 β-HSD, LH-R;
(4) karyotyping, this clone do not take place chromosomally to lose or increase, and caryogram is stable;
(5) show that with external tumorigenicity test this clone does not have the conversion characteristic of tumour in the body.
Description of drawings
Fig. 1 is the form of transfection front and back goat lutein cell under the inverted microscope: A: former generation goat lutein cell (100 *); B: 15 generation goat lutein cells (hTERT-GLCs) (100 *) after the transfection; C: 30 generation goat lutein cells (100 *) after the transfection; D: 55 generation goat lutein cells (100 *) after the transfection;
The goat lutein cell is identified in the dyeing of Fig. 2 histocytochemistry: steroid dehydrogenase dyeing evaluation (show blue around the nucleus of positive cell, 100 *);
Goat lutein cell growth curve before and after Fig. 3 transfection: goat lutein cell (hTERT-GLCs) and goat lutein cell of former generation in the 2nd generation (GLCs) after the 55th generation after the transfection;
Fig. 4 RT-PCR detects the expression of transfection front and back goat lutein cell human telomerase reverse transcriptase gene mRNA: in goat lutein cell the 30th generation (swimming lane 2) and the 55th generation (swimming lane 3) after the transfection,, former generation goat lutein cell the 2nd generation (swimming lane 4) and HeLa cell (swimming lane 1) are as positive control;
Goat lutein cell telomerase activation after the transfection of Fig. 5 telomerase activation detection kit (Roche) mensuration;
1: cervical cancer cell (HeLa), 2: the 30th generation of goat lutein cell after the transfection, 3: goat lutein cell after the 55th generation after the transfection, former generation in 4: the 2 generations the goat lutein cell
Fig. 6 RT-PCR detects the expression of transfection front and back goat lutein cell key gene mRNA;
Fig. 7 radioimmunoassay cyclic adenosine monophosphate (cAMP) (8-Br-cAMP), (22 (R)-hydroxycholesterol) and Vitarrine (Pregnenolone) are to the influence of goat lutein cell progesterone secretion amount after the transfection for 22 (R)-oxycholesterols;
Goat lutein cell karyotyping result after Fig. 8 transfection;
Fig. 9 soft agar test-results A:HeLa cell is cultivated 3 weeks (positive control), B on soft agar: the goat lutein cell was cultivated for 3 weeks on soft agar after the transfection;
Figure 10 tumor after being inoculated into nude mice test-results A: nude inoculation HeLa cell is inoculated district's weave construction (HE, 100 *) after February, and the tissue slice inspection is found, the cell arrangement disorder, and loss of polarity is soaked into to the deep, and visible a large amount of nuclear fission phases present the feature of malignant tumour.B: the goat lutein cell is inoculated district's weave construction (HE, 100 *) after nude inoculation the 55th generation transfection after February, and the good and healthy tissues of weave construction does not have tangible difference than seeing.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
One. cell cultures
1. the separation of goat lutein cell and purifying
Get the ovary (judging the Gestation period) of the healthy Sa energy milk goat in pregnant 6-8 week by checking fetus size and observation development degree in local slaughterhouse, being put into 4 ℃ is added with in two anti-phosphate buffered saline buffers (PBS), take back in the 30min in the super clean bench of laboratory, peel off luteal tissue and be cut into 1mm as far as possible
3Organize fragment, add 0.2% (mass volume ratio) collagenase II 2ml, after putting in 37 ℃ of thermostat water baths digestion luteal tissue 15min, add and contain 15% foetal calf serum, 100IU/mL penicillin, the DMEM/F12 of 100 μ g/mL Streptomycin sulphates (Dulbecco ' s Modified Eagle ' s andHAM ' S F-12,1: 1 (v/v) with 15mmol/I HEPES) substratum, blow to cell dispersion, and filter with 150 μ m nylon membranes, remove and do not digest tissue block completely, collecting filtered liquid carries out centrifugal and repeated washing 3 times, with the DMEM/F12 substratum suspension precipitation that contains 15% foetal calf serum, repel test with trypan blue and detect cell activity, and the adjustment cell density makes viable cell reach 1 * 10 then
6Individual/ml, be inoculated in 25cm
2In the culturing bottle, be positioned over 37 ℃, 5%CO
2Cultivate in the incubator.Change liquid after 3 days, treat cell cover with bottle at the bottom of the time go down to posterity, carry out the differential digestion purifying with citric acid-trypsinase, and according to the histocytochemistry coloration result, calculate the ratio of the shared total cellular score of positive cell at microscopically, treat that lutein cell purity reaches 95% and carries out follow-up transfection when above.
2. lutein cell is identified in histocytochemistry dyeing
(active coloring of 3 β-HSD) carries out the evaluation of steroid lutein cell, and lutein cell dyeing back is in nucleus peripheral layer mazarine by 3beta-Hydroxysteroid dehydrogenase in the pair cell.Cultured cells is inoculated in 6 orifice plates, removes substratum behind the 24h, and washes twice with PBS, and the formaldehyde fixed 20min with 1% adds dye liquor then and (contains in the PBS liquid of 0.1M; 0.1%BSA, 1.5mM NAD, 0.25mM chlorination nitro tetrazole and 0.2mM dehydroepiandrosterone), place 4h 37 ℃ of lucifuges, wash twice with PBS liquid then, mirror is observed the (see figure 2) of taking pictures down.
3 transfections and screening
Lutein cell 0.25% trysinization of former generation in the 2nd generation does not stop digestion and is inoculated into six orifice plates with not containing two anti-substratum.Treat that cell reaches when converging more than 90%, remove substratum, and with the basic medium washed twice that does not contain serum.Liposome 2000 specification sheetss according to Invitrogen company carry out transfection, with liposome 2000 human telomerase reverse transcription gene is imported in the lutein cell, add (450 μ g/ml) G418 after the transfection and screen 2-3 week, after forming Deng mono-clonal, the picking mono-clonal is identified lutein cell with the dyeing of step 2 histocytochemistry.
4 positive cell enlarged culturing: the positive cell that histocytochemistry dyeing is identified is inoculated into 25cm
2In the Tissue Culture Flask, G418 keeps cultivation with lower concentration, goes down to posterity once every 3 days, divides kind of rate 1: 2, externally carries out the inspection and the checking of biological indicator when reaching for 30 generations.
The mensuration of the growth curve of goat lutein cell before and after 5 transfections
With the 2nd generation the goat lutein cell and the transfection of the 55th generation after the goat lutein cell by 1 * 10
4Individual cells/well is inoculated in 24 well culture plates, and 3d changes liquid 1 time, uses 0.25% trypsin digestion and cell every day, the blood cell counting plate counting.Each hole counting 3 times, get 3 holes at every turn, get its mean value and draw the growth curve of cell, and calculate population doubling time (PDT): PDT=[log2/ (LogNt-logN0) as follows] * t, wherein N0 and Nt represent when inoculating respectively and the cell count (see figure 3) of cultivation after t hour.
The graceful enzyme expression of gene of goat lutein cell after the 6RT-PCR detection transfection
From the 30th generation, in goat lutein cell and 2nd generation goat lutein cell extract total mRNA after the transfection of 55 generations with reference to the operation instructions of the Trizol Reagent of Invitrogen company, and carry out reverse transcription and become cDNA, carry out pcr amplification, amplification condition then; First at 95 ℃ of following sex change 3min, 95 ℃ then, 30s, 56 ℃, 40s, 72 ℃, 60s, 35 circulations are extended 10min at 72 ℃ at last.Get above-mentioned PCR product 6 μ L electrophoresis in 0.2% sepharose, ethidium bromide colour developing after 120V, 30min electrophoresis finish is in the observation of ultraviolet gel imaging system, the (see figure 4) of taking pictures.
7 telomerase activations detect
According to test kit TeloTAGGGTelomerasePCRELISA
PLUSOperation instructions among the kit (Roche) is carried out: Telomerase extracts, and Telomerase extends and pcr amplification reaction, and ELISA measures.Collect 2 * 10
5Individual cell, with Lysis buffer at cracking 30min on ice.12000rpm, 20min gets 1 μ l cracking supernatant liquor as Telomerase template, increases according to the pcr amplification reaction condition of test kit, and negative control is handled with RNAase.After amplification finishes, develop the color and the absorbance measurement (see figure 5) by ELISA requirement and application of sample order.
The expression of key gene in the goat lutein cell after the 8RT-PCR detection transfection
The cDNA that utilizes in the step 6 mRNA that extracts to be transcribed into carries out PCR, and the pcr amplification reaction condition is the same with step 3, carries out gel electrophoresis then, the imaging and the (see figure 6) of taking pictures.
9 radioimmunities detect the progesterone secretion of transfection goat lutein cell
According to iodine [
125I] specification sheets of progesterone radioimmunoassay medicine box (Tianjin Jiuding Medical Biological Engineering Co., Ltd) carries out: sample is prepared and is handled.To reach the 55th generation the goat lutein cell be inoculated in 12 well culture plates, 24h removes substratum, and add the cyclic adenosine monophosphate (cAMP) analogue contain different concns respectively, 22-oxycholesterol and Vitarrine, each concentration repeats 3 holes, and 24h collects supernatant, 4 ℃, 3500r/min is centrifugal, and 15min gets supernatant, and it is to be measured to put-20 ℃ of preservations.According to given typical curve, calculate progesterone levels (see figure 7) in the testing sample.
10 karyotypings
The cell in vegetative period of taking the logarithm adds colchicine and continues to cultivate 6h to concentration 0.1 μ g/mL, uses 0.25% trypsin digestion and cell, PBS liquid washed cell 1 time, and the centrifugal 5min of 800r/min abandons supernatant.With 75mmol/L KCl re-suspended cell, room temperature is placed 10min, some methyl alcohol/Glacial acetic acid (v/v 3: 1) stationary liquid then, behind the mixing 4 ℃ centrifugal, abandon supernatant, continue to add stationary liquid, place 30min for 4 ℃, 4 ℃ centrifugal, abandons supernatant, how much add an amount of stationary liquid re-suspended cell according to cell, the back drips on the slide of 4 ℃ of precoolings, and air rapid drying is with about Ji's nurse Sa dyeing 10min, conventional dehydration mounting, oily mirror are observed (see figure 8) down.
11 soft agar suspension tests
Spread earlier and contain 0.35% agar, get goat lutein cell after the transfection of the 55th generation, be prepared into single cell suspension, and be diluted to 2000 cell/ml, mix by 1: 1 (volume) with 1.2% agar then, as upper strata glue at 12 well culture plate bottoms.Phase microscope is observed clone's formation situation down after cultivating for 3 weeks.The positive contrast of HeLa cell (the results are shown in Figure 9).
The test of 12 tumor after being inoculated into nude mice
Collect and go up the sample lutein cell after the transfection of the 55th generation, resuspended and to adjust cell density be 1 * 106/ml with substratum, in the subcutaneous aseptic inoculation cell suspension of 6 all nude mice buttocks in age 0.1mL, routine observation.The positive contrast of HeLa cell.The bimester after nude mice dislocation cause death, observe tumour and have or not and inoculation position is carried out tissue slice inspection (HE dyeing) (the results are shown in Figure 10).
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (3)
1. the establishment method of a goat lutein cell system is characterized in that, may further comprise the steps:
(1) former being commissioned to train supported and purifying: the healthy Sa energy milk goat corpus luteum of getting pregnant 6-8 week, aseptic condition is the digestion luteal tissue down, and a lutein cell that obtains is inoculated in the Tissue Culture Flask, cultivate with DMEM/F12, described DMEM/F12 contains 15% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates; Being juxtaposed to 37 ℃, 5%CO2 incubator cultivates; Go down to posterity after covering with culturing bottle in cell, carry out purifying with citric acid-trypsinase differential digestion;
(2) transfection and screening: former generation lutein cell that pair cell purity reaches more than 95% utilizes liposome transfection human telomerase reverse transcriptase gene, and screens 2-3 week with G418, and the picking mono-clonal is also identified with histocytochemistry dyeing;
(3) enlarged culturing: positive cell is carried out enlarged culturing, went down to posterity once in 3 days, divided kind of rate 1: 2, treat to carry out when cell passed to for 30 generations qualification test, and measure the growth curve of transfection front and back goat lutein cell.
2. by the described method of claim 1, it is characterized in that, described step (2) is identified the method for lutein cell: by 3beta-Hydroxysteroid dehydrogenase active coloring in the pair cell, carry out the evaluation of steroid lutein cell, lutein cell dyeing back is in nucleus peripheral layer mazarine.
3. by the described method of claim 1, it is characterized in that described step (2) is screened with 450 μ g/ml G418.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391987A (en) * | 2011-11-17 | 2012-03-28 | 安徽农业大学 | Digestive juice for separating mouse fat stem cells |
| CN110669720A (en) * | 2019-09-27 | 2020-01-10 | 中国农业大学 | Method for improving in-vitro primary yellow body cell function of pig |
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| CN1336431A (en) * | 2001-08-23 | 2002-02-20 | 北京大学人民医院 | Establishment of immortal human ovary carcinoma cell strain |
| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1336431A (en) * | 2001-08-23 | 2002-02-20 | 北京大学人民医院 | Establishment of immortal human ovary carcinoma cell strain |
| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
Non-Patent Citations (2)
| Title |
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| 《中国优秀硕士学位论文全文数据库（农业科技辑）》 20101115 杨艳 hTERT 基因转染建立永生化山羊卵巢间质细胞的研究 摘要、第19页第3.1－3.4节、第27页第4.2.2节、第32页第4.3.4.1节 1－3 , 第11期 * |
| 《西北农林科技大学学报(自然科学版)》 20091130 权富生等 山羊卵巢间质细胞分离方法的比较 全文 1－3 第37卷, 第11期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102391987A (en) * | 2011-11-17 | 2012-03-28 | 安徽农业大学 | Digestive juice for separating mouse fat stem cells |
| CN110669720A (en) * | 2019-09-27 | 2020-01-10 | 中国农业大学 | Method for improving in-vitro primary yellow body cell function of pig |
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Application publication date: 20110810 |