CN1291011C - In vitro culturing method of oviduct secretory cell of Chinese forest frog - Google Patents
In vitro culturing method of oviduct secretory cell of Chinese forest frog Download PDFInfo
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- CN1291011C CN1291011C CN 200510016615 CN200510016615A CN1291011C CN 1291011 C CN1291011 C CN 1291011C CN 200510016615 CN200510016615 CN 200510016615 CN 200510016615 A CN200510016615 A CN 200510016615A CN 1291011 C CN1291011 C CN 1291011C
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Abstract
The present invention relates to an in vitro culture method for secretory cells of the oviducts of Rana temporaria chensinensis, which belongs to an in vitro culture method for secretory cells of the oviducts of economic animals. The present invention comprises the following steps: the preparation of culture media for secretory cells of the oviducts, the obtainment of primary cells secreted by the oviducts, the culture of the primary cells secreted by the oviducts, and the passage culture of the secretory cells of the oviducts, wherein the preparation of the culture media for secretory cells of the oviducts comprises the preparation of a basic culture medium and a conditional culture medium. In view of the special medical value and the wide application prospect of the oviducts of Rana temporaria chensinensis, the present invention has the advantages that a conditional culture medium for the in vitro culture of the secretory cells of the oviducts of Rana temporaria chensinensis is determined, the primary culture and the passage culture of the secretory cells of the oviducts of Rana temporaria chensinensis in vitro are realized, secretory cells of the oviducts of Rana temporaria chensinensis, which have distinct biological functions, are cultured method by a cell engineering method and other biological technology methods, and Rana temporaria chensinensis as natural resources can be preferably developed, applied and protected.
Description
Technical field
The invention belongs to the oviduct secretory cell cultural method of a kind of economic animal, especially cultivate at this kind cells in vitro.
Background technology
Rana temporaria chensinensis David (Rana chensinensis David) is commonly called as " Oviductus Ranae ", it is a kind of important medicinal batrachians economic animal, be distributed widely in areas such as Changbai Mountain, the northeast arteries and veins and the Xiaoxinanlin Mountains, the uterine tube dry products of the female frog promptly is well-known " dried oviduct fat of the forest frog ", be famous and precious dietotherapy Chinese medicine, include multiple saturated fatty acid, amino acid, VITAMIN and trace element, have Yin nourishing and lung moistening, tonifying kidney and benefiting sperm, effects such as brain tonic antifatigue.In the last few years, because a large amount of the seizure, the resource of Rana temporaria chensinensis David was seriously damaged, and also supply falls short of demand as the needs of medicinal raw material with it, can not satisfy and the requirement of domestic and international market.
Summary of the invention
The invention provides a kind of Rana temporaria chensinensis David oviduct secretory cell extracorporeal culturing method, solving at present in order to obtain the uterine tube dry products of the female frog, and the problem that the resource of Rana temporaria chensinensis David is seriously damaged.The technical scheme that the present invention takes is:
One, the preparation of the substratum of oviduct secretory cell:
To the basic medium that is fit to the Rana temporaria chensinensis David oviduct secretory cell DMEM/Ham ' sF12, F10/DMEM, RPMI1640 are arranged by experiment sieving, every part of above-mentioned substratum is dissolved in 1000ml four to be heated up in a steamer in the water, add microbiotic wherein penicillin 100,000 units/L, Streptomycin sulphate 100mg/L again, substratum is with 0.22 μ m filter membrane Entkeimung, and adjusting pH is that 7.4-7.6 is standby;
Conditioned medium comprises that as the serum of nutritive substance and the growth-promoting class hormone of interpolation, wherein new-born calf serum is to add 5～15ml in every 100mL basic medium, and Regular Insulin is 5～10mg/L basic medium; 10
-6The estradiol of mol/L is 5～10 μ l/ml basic mediums;
Two, obtaining of uterine tube secretion primary cell:
The aseptic uterine tube of taking adopts direct shear method dissociated cell, thereby reduces the degree of injury of pair cell and provide better precondition for external survival;
The present invention adopts an important embodiment case of direct shear method to be: destroy myelencephalon with dissecting needle and put to death wood frog, in gnotobasis, cut Rana temporaria chensinensis David female the preovulatory phase ampulla of uterine tube as for the sterilization little plate in, float uterine tube 2～3 times with D-Hank ' the s liquid that contains mycillin, separate behind the flush away blood stains, cut off adhesion organization on every side, in plate, uterine tube is vertically cut off, the uterine tube of cutting off is further shredded, 800r/min is centrifugal 10 minutes after gauze filters, abandon supernatant, the precipitation part is used the basic culture solution rinsing again, eccentric cell 2 times is abandoned supernatant liquor and is obtained wood frog oviduct secretion primary cell;
Three, the cultivation of uterine tube secretion primary cell:
To obtain to such an extent that primary cell adds and to contain 15% serum nutrient solution and make cell suspension, through the cell counting of trypan blue dyeing row, adjusting cell concn is 2 * 10
5～5 * 10
5Individual/ml is inoculated in the 6 hole culture dish, and every hole adds cell suspension 3ml, puts into 37 ℃, the incubator of 5%CO2 and cultivates.Changed one time nutrient solution in per 3～4 days.
Four, the cultivation of going down to posterity of oviduct secretory cell:
After treating that primary cell covers with individual layer, can go down to posterity, inhale and remove original fluid in the culture dish, use basic culture solution washed cell 1-2 time, the serum composition that flush away is residual.Add 0.125%-0.25% trypsinase 2ml, put into 37 ℃ of incubator digestion 5-10 minute, inverted microscope is observed attached cell contraction change circle down can inhale digestive ferment liquid, add and contain 15% serum stop buffer termination digestion, abandon stop buffer, add basic culture solution, cell suspension is made in piping and druming, and regulating cell count is 10
5～2 * 10
5Individual/ml is inoculated in the new culture dish.Change liquid according to the every 2-3 of cell growing state days, note staying 1/4 original fluid when changing liquid, finish once and go down to posterity.
By test determine the wood frog oviduct secretory cell go down to posterity that cultivate should be in 3-4 generation.
The invention has the beneficial effects as follows at oviducal special pharmaceutical use of Rana temporaria chensinensis David and application prospects, the present invention is to the oviduct secretory cell of Rana temporaria chensinensis David, carry out former generation and go down to posterity cultivation, and to biological characteristic researches such as its form and growing multiplication abilities, successfully find out oviduct secretory cell vitro culture technology, for further studying the growth of wood frog oviduct secretory cell from now on, metabolic differentiation, various hormones, cytokine provides the ideal experimental model to its effect, lays the foundation for utilizing cell engineering to produce wood frog fallopian tube secretions matter from now on.Seek out the separation method that is fit to the wood frog oviduct secretory cell by the present invention, cell is separated from tissue; Select the substratum of suitable wood frog oviduct secretory cell to be used for cultivating, and found out the adding conditional of some nutritive substances, determined the conditioned medium of wood frog oviduct secretory cell vitro culture; Realized that external former being commissioned to train of wood frog oviduct secretory cell support and cultivations of going down to posterity, and method such as employing immunohistochemical methods is identified to the cell of gained.
Beneficial effect of the present invention also comprises; utilize biotechnological meanss such as cell engineering; has unique biological function wood frog oviduct secretory cell in vitro culture; for technical foundation is established in a large amount of and acquisition " the artificial dried oviduct fat of the forest frog " in the future; be in order to develop, use and protect the natural resource of Rana temporaria chensinensis David better, for the protection eubiosis plays a driving role
Description of drawings
Fig. 1 is the primary cell photo of wood frog oviduct secretory cell, just dissociated state 10 * 10.
Fig. 2 is the primary cell photo of wood frog oviduct secretory cell, cultivates state 10 * 10 after 24 hours.
Fig. 3 is the primary cell photo of wood frog oviduct secretory cell, just dissociated state 25 * 10.
Fig. 4 is the primary cell photo of wood frog oviduct secretory cell, cultivates state 25 * 10 after 24 hours.
Fig. 5 is the passage cell photo of wood frog oviduct secretory cell, cultivates state 10 * 10 after 24 hours.
Fig. 6 is the immunohistochemical methods photo of wood frog oviduct secretory cell, and endochylema pale brown look, karyon mazarine 10 * 10.
Embodiment
Embodiment 1
Material and instrument
Laboratory animal
Selecting body weight for use is 0.1-0.15kg, and the female Rana temporaria chensinensis David before laying eggs is as the oviduct secretory cell donor.Reagent
Trypsin 1: 250), D-Hank ' s, RPMI1640 substratum are available from U.S. Gibco company; New-born calf serum (NBS), trypan blue are Sigma company product, and penicillin and streptomycin etc. are homemade conventional reagent.Instrument
MCO-17AI type CO
2Incubator (Japan, SANYO company); CQIC type inverted microscope (optical instrument factory, Chongqing);
SW-CJ-IF type Bechtop; Tissue Culture Plate (Denmark NUCO company).
One. wood frog oviduct secretory cell culture medium preparation
The RPMI1640 substratum of a standard pack is dissolved in 1000ml four surely heats up in a steamer in the water, add microbiotic again, wherein penicillin 100,000 units/L, Streptomycin sulphate 100mg/L.Fully substratum adjustment pH is 7.4 behind the mixing, with standby after the 0.22 μ m filter membrane Entkeimung,
The preparation of wood frog oviduct secretory cell conditioned medium
Be basic culture solution with RPMI-1640, adding this volume 5% calf serum, Regular Insulin 5mg/L, concentration is 10
-6The estradiol 10 μ l/ml of mol/L form conditioned medium.
Two. obtaining of wood frog oviduct secretion primary cell
Destroy myelencephalon with dissecting needle and put to death wood frog, frog body and function 75% is alcohol-pickled in gnotobasis, the taking-up uterine tube of cutting open the belly, thick tortuous more ampulla in the middle of cutting, drop at once after the excision and fill 100IU/ml penicillin, in the low temperature D-hanks liquid of 100 μ g/ml Streptomycin sulphates, rinsing uterine tube 2～3 times, in plate, uterine tube is vertically cut off behind the flush away blood stains, the uterine tube of cutting off is further shredded, adding basic medium purge is repeatedly organized fragment, draw the basic medium after washing, after gauze filtered, centrifugal 10 minutes of 1000r/min abandoned supernatant, the precipitation part is used the basic medium rinsing again, eccentric cell 2 times obtains wood frog oviduct secretion primary cell.(see figure 1)
Three. the cultivation of wood frog oviduct secretion primary cell
To obtain to such an extent that primary cell adds and to contain 15% serum nutrient solution and make cell suspension.Through the cell counting of trypan blue dyeing row, adjusting cell concn is 2 * 10
5Individual/ml is inoculated in the 6 hole culture dish (Denmark produces Nunc), and every hole adds cell suspension 3ml, puts into 37 ℃, 5%CO
2Incubator in cultivate.Changed one time the nutrient solution (see figure 2) in per 3～4 days.Cultivate always and do not go down to posterity, primary cell can be survived about 20 days.
Four. the cultivation of going down to posterity of wood frog oviduct secretory cell
After treating that primary cell grows up to individual layer, promptly go down to posterity.Original fluid in the culture dish is removed in suction, with basic medium washed cell 1 time, the serum composition that flush away is residual, every hole add 0.125% trypsinase 2ml, put into 37 ℃ of incubator digestion 10 minutes, inverted microscope is observed round promptly suction of attached cell contraction change down and is removed digestive ferment liquid, add and contain the termination digestion in 2 minutes of 15% serum basic culture solution, abandon nutrient solution, add basic culture solution, cell suspension is made in piping and druming, and regulating cell count is 2 * 10
5Individual/ml is inoculated in the new culture dish, finishes the process that once goes down to posterity, and goes down to posterity to cultivate to carry out for 3 generations altogether.
Embodiment 2
Material and instrument
Laboratory animal
Selecting body weight for use is 0.1-0.15kg, and the female Rana temporaria chensinensis David before laying eggs is as the oviduct secretory cell donor.Reagent
Disodium ethylene diamine tetraacetate (EDTA), D-Hank ' s, DMEM/Ham ' sF12 substratum are available from U.S. Gibco company; New-born calf serum (NBS), trypan blue are Sigma company product, and penicillin and streptomycin etc. are homemade conventional reagent.
Instrument
MCO-17AI type CO
2Incubator (Japan, SANYO company); CQIC type inverted microscope (optical instrument factory, Chongqing);
SW-CJ-IF type Bechtop; Tissue Culture Plate (Denmark NUCO company).
One. wood frog oviduct secretory cell culture medium preparation
DMEM/Ham ' the sF12 substratum of a standard pack is dissolved in 1000ml four surely heats up in a steamer in the water, add microbiotic again, wherein penicillin 100,000 units/L, Streptomycin sulphate 100mg/L.Fully substratum adjustment pH is 7.5 behind the mixing, with standby after the 0.22 μ m filter membrane Entkeimung.
The preparation of wood frog oviduct secretory cell conditioned medium
Be basic culture solution with DMEM/Ham ' sF12, adding its volume 10% calf serum, Regular Insulin 8mg/L, concentration is 10
-6The estradiol 7 μ l/ml of mol/L form conditioned medium.
Two. obtaining of wood frog oviduct secretion primary cell
Destroy myelencephalon with dissecting needle and put to death wood frog, frog body and function 75% is alcohol-pickled in gnotobasis, the taking-up uterine tube of cutting open the belly, thick tortuous more ampulla in the middle of cutting, drop at once and fill in the low temperature D-hanks liquid of 100IU/ml penicillin 100 μ g/ml Streptomycin sulphates, rinsing uterine tube 2～3 times, in plate, uterine tube is vertically cut off behind the flush away blood stains, the uterine tube of cutting off is further shredded, adding basic medium purge is repeatedly organized fragment, draw the basic medium after washing, after gauze filtered, centrifugal 10 minutes of 1100r/min abandoned supernatant, the precipitation part is used the basic medium rinsing again, eccentric cell 2 times obtains wood frog oviduct secretion primary cell.
Three. the cultivation of wood frog oviduct secretion primary cell
To obtain to such an extent that primary cell adds and to contain 15% serum nutrient solution and make cell suspension.Through the cell counting of trypan blue dyeing row, adjusting cell concn is 3 * 10
5Individual/ml is inoculated in the 6 hole culture dish (Denmark produces Nunc), and every hole adds cell suspension 3ml, puts into 37 ℃, 5%CO
2Incubator in cultivate.Changed one time nutrient solution in per 3～4 days, cultivate always and do not go down to posterity, primary cell can be survived about 20 days.
Four. the cultivation of going down to posterity of wood frog oviduct secretory cell
After treating that primary cell grows up to individual layer, promptly go down to posterity.Original fluid in the culture dish is removed in suction, uses basic medium washed cell 1 time, the serum composition that flush away is residual.Every hole adds cell dissociation agent 0.02%EDTA-Na 3ml, puts into 37 ℃ of incubator digestion 10 minutes, and inverted microscope is observed the attached cell contraction down and become circle, collects cell suspension, adds in the centrifuge tube centrifugal 10 minutes of 1000r/min.Abandon supernatant, add basic culture solution washing 2 times, abandon the washing basic culture solution, add the conditioned medium re-suspended cell, cell suspension is made in piping and druming gently, and regulating cell count is 10
5Individual/ml is inoculated in enlarged culturing in the new culture dish, once goes down to posterity, and goes down to posterity to cultivate to carry out for 4 generations altogether.
Embodiment 3
Material and instrument
Laboratory animal
Selecting body weight for use is 0.1-0.15kg, and the female Rana temporaria chensinensis David before laying eggs is as the oviduct secretory cell donor.Reagent
Trypsinase, D-Hank ' s, F10/DMEM substratum are available from U.S. Gibco company; New-born calf serum (NBS), trypan blue are Sigma company product, and penicillin and streptomycin etc. are homemade conventional reagent.
Instrument
MCO-17AI type CO
2Incubator (Japan, SANYO company); CQIC type inverted microscope (optical instrument factory, Chongqing);
SW-CJ-IF type Bechtop; Tissue Culture Plate (Denmark NUCO company).
One. wood frog oviduct secretory cell culture medium preparation
The F10/DMEM substratum of a standard pack is dissolved in 1000ml four surely heats up in a steamer in the water, add microbiotic again, wherein penicillin 100,000 units/L, Streptomycin sulphate 100mg/L.Fully substratum adjustment pH is 7.6 behind the mixing, with standby after the 0.22 μ m filter membrane Entkeimung.
The preparation of wood frog oviduct secretory cell conditioned medium
Be basic culture solution with F10/DMEM, adding its volume 15% calf serum, Regular Insulin 10mg/L, concentration is 10
-6The estradiol 5 μ l/ml of mol/L form conditioned medium.
Two. obtaining of wood frog oviduct secretion primary cell
Destroy myelencephalon with dissecting needle and put to death wood frog, frog body and function 75% is alcohol-pickled in gnotobasis, the taking-up uterine tube of cutting open the belly, thick tortuous more ampulla in the middle of cutting, drop at once and fill in the low temperature D-hanks liquid of 100IU/ml penicillin 100 μ g/ml Streptomycin sulphates, rinsing uterine tube 2～3 times, in plate, uterine tube is vertically cut off behind the flush away blood stains, the uterine tube of cutting off is further shredded, adding basic medium purge is repeatedly organized fragment, draw the basic medium after washing, after gauze filtered, centrifugal 10 minutes of 1200r/min abandoned supernatant, the precipitation part is used the basic medium rinsing again, eccentric cell 2 times obtains wood frog oviduct secretion primary cell.
Three. the cultivation of wood frog oviduct secretion primary cell
To obtain to such an extent that primary cell adds and to contain 15% serum nutrient solution and make cell suspension.Through the cell counting of trypan blue dyeing row, adjusting cell concn is 5 * 10
5Individual/ml is inoculated in the 6 hole culture dish (Denmark produces Nunc), and every hole adds cell suspension 3ml, puts into 37 ℃, 5%CO
2Incubator in cultivate.Changed one time nutrient solution in per 3～4 days.Cultivate always and do not go down to posterity, primary cell can be survived about 20 days.
Four. the cultivation of going down to posterity of wood frog oviduct secretory cell
After treating that primary cell grows up to individual layer, promptly go down to posterity.Original fluid in the culture dish is removed in suction, uses basic medium washed cell 1 time, the serum composition that flush away is residual.Every hole adds 0.25% trypsinase 2ml, puts into 37 ℃ of incubator digestion 5 minutes, and inverted microscope is observed attached cell down and shunk the change circle, collects cell suspension, adds in the centrifuge tube centrifugal 10 minutes of 1000r/min.Abandon supernatant, add basic culture solution washing 2 times, abandon the washing basic culture solution, add the conditioned medium re-suspended cell, cell suspension is made in piping and druming gently, and regulating cell count is 2 * 10
5Individual/ml is inoculated in enlarged culturing in the new culture dish, once goes down to posterity, and goes down to posterity to cultivate to carry out for 3 generations altogether.
Experimental example
1, the observation of wood frog oviduct secretory cell
As seen, just isolating wood frog oviduct secretion primary cell is an oval under the inverted microscope,, endochylema is full, and the nuclear circle is big, is the suspended state (see figure 3).Cell inoculation is mostly adherent after 24 hours, stretches to be olive shape, similar eyes shape (see figure 4).The adherent stretching, extension of passage cell is fast, and about 4～5d merges into the individual layer (see figure 5), and along with passage number increases, cell gradually becomes the spindle shape cell.
2, the evaluation of wood frog oviduct secretory cell
CD34 is a kind of endothelial cell adhesion molecule, and its structure contactin member is a kind of surface marker of endotheliocyte.Anti-CD34 monoclonal antibody is all stronger to endotheliocyte stability and susceptibility.With anti-CD34 monoclonal antibody immunity group test kit.Undertaken by the explanation in the test kit by the immunohistochemical staining method, the immunocytochemical stain result shows that the oviduct secretory cell positive is painted, and other cell tissues all are unstained.Oviduct secretory cell antagonism CD34 monoclonal antibody is positive, and cytoplasm is dyed brown or pale brown look, and nucleus the mazarine (see figure 6), presents (interior) Pi Yuanxing.
Claims (2)
1, a kind of Rana temporaria chensinensis David oviduct secretory cell extracorporeal culturing method comprises the following steps:
One, the preparation of the substratum of oviduct secretory cell: comprise basic medium, conditioned medium;
The basic medium of selecting to be fit to the Rana temporaria chensinensis David oviduct secretory cell has DMEM/Ham ' sF12, F10/DMEM or RPMI1640, every part of above-mentioned substratum is dissolved in the 1000ml quadruple distillation water surely, add microbiotic again, wherein penicillin 100,000 units/L, Streptomycin sulphate 100mg/L, with 0.22 μ m filter membrane Entkeimung, adjustment pH is 7.4-7.6;
Conditioned medium comprises adding 5～15ml new-born calf serum as the new-born calf serum of nutritive substance and the growth-promoting class hormone of interpolation in the above-mentioned basic medium of wherein every 100mL that Regular Insulin is 5～10mg/L basic medium; 10
-6The estradiol of mol/L is 5～10 μ l/ml basic mediums, is made into conditioned medium;
Two, obtaining of uterine tube secretion primary cell:
The aseptic uterine tube of taking adopts direct shear method dissociated cell, thereby reduces the degree of injury of pair cell and provide precondition for external survival;
Three, the cultivation of uterine tube secretion primary cell:
The primary cell that obtains is added the substratum that contains 15% bovine serum make cell suspension, through trypan blue dyeing carrying out cell counting, adjusting cell concn is 2 * 10
5～5 * 10
5Individual/ml is inoculated in the 6 hole culture dish, and every hole adds cell suspension 3ml, puts into 37 ℃, the incubator of 5%CO2 and cultivates, and changes a subculture in per 3～4 days;
Four, the cultivation of going down to posterity of oviduct secretory cell:
After treating that primary cell covers with individual layer, promptly go down to posterity, culture dish Central Plains substratum is removed in suction, with basic medium washed cell 1-2 time, the serum composition that flush away is residual, add 0.125% or 0.25% trypsinase 2ml or 0.02% disodium ethylene diamine tetraacetate 3ml, put into 37 ℃ of incubator digestion 5-10 minute, inverted microscope is observed attached cell down and is shunk the change circle, inhales and goes added trypsinase or 0.02% disodium ethylene diamine tetraacetate, adds to contain the digestion of 15% serum stop buffer, abandon this stop buffer, add basic medium or conditioned medium, cell suspension is made in piping and druming, and regulating cell count is 10
5～2 * 10
5Individual/ml is inoculated in the new culture dish, finishes once and goes down to posterity.
2, Rana temporaria chensinensis David oviduct secretory cell extracorporeal culturing method according to claim 1 is characterized in that: in going down to posterity of step 4 oviduct secretory cell cultivated, go down to posterity and cultivate the generation at 3-4.
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| CN 200510016615 CN1291011C (en) | 2005-03-09 | 2005-03-09 | In vitro culturing method of oviduct secretory cell of Chinese forest frog |
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| CN 200510016615 CN1291011C (en) | 2005-03-09 | 2005-03-09 | In vitro culturing method of oviduct secretory cell of Chinese forest frog |
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| CN1291011C true CN1291011C (en) | 2006-12-20 |
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Effective date of registration: 20070928 Address after: Xincheng Street Jingyue District in Jilin province 130118 Changchun No. 2888 Patentee after: Jilin Agricultural University Address before: Xincheng Street Jingyue District in Jilin province 130118 Changchun No. 2888 Co-patentee before: Zheng Xin Patentee before: Liu Jingsheng |
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