CN106906177A - A kind of naked mole interstitial glands is isolated and purified and cultural method - Google Patents
A kind of naked mole interstitial glands is isolated and purified and cultural method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0683—Cells of the male genital tract, e.g. prostate, epididymis; Non-germinal cells from testis, e.g. Leydig cells, Sertoli cells
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The present invention relates to field of cell culture, specifically a kind of isolation and purification method of naked mole interstitial glands isolates and purifies naked mole interstitial glands using enzymic digestion and Percoll gradient of continuous density methods.The inventive method being capable of easy, efficiently, the economic normal naked mole interstitial glands of a large amount of functional activities of acquisition, culture under hypoxia condition ensure that the biological characteristics that this cell can still be maintained under body state in ex vivo environment, consequently facilitating the special physiological function of naked mole interstitial glands is directly further studied in pure in-vitro cell culture model, so as to provide important theoretical foundation to explore biological mechanism therein and being applied to clinical relevant.
Description
Technical field
It is that a kind of naked mole interstitial glands is separated, purified specifically the present invention relates to technical field of cell culture
And cultural method.
Background technology
Interstitial glands (Leydig cell) is distributed in the loose connective tissue between testicular spermatogenic tubule, accounts for institute
Have the 2%~4% of testis tissue inner cell quantity, its major function is synthesis and Testosterone Secretion, the testosterone of its secretion accounts for blood plasma
95% (Dobashi M, Fujisawa M, Yamazaki T, et a1.Inhibition of of testosterone total amount
steroidogenesis in Leydig cells by exogenous nitric oxide Occurs
independently of steroidogenic acute regulatory protein(star)mRNA[J].Arch
Androl, 2001,47 (3)：203-209.).In view of the important function that interstitial glands has, sets up dividing for a set of stabilization
From, purifying and the method for original cuiture interstitial cell plastid, and the activity of the cell Testosterone Secretion is kept, for further carrying out testis
The research of ball interstitial cell correlation function is significant.
Naked mole is a kind of eusociality rodent for originating in East Africa some areas, and its fecundity can be in length
Keep invariable up in the life-spans of 30 years, size of animal can reach more than 300 (Braude S.Dispersal in group
and new colony formation in wild naked molerats:evidence against inbreeding
as the system of mating[J].Behav Ecol.2000(11):7-12.).No matter in the wild naturally growth or
Under the conditions of laboratory is raised and train, the fecundity of naked mole is only defined in a raettin and 3-5 participation that specially department breeds and breeds
Male mouse, remaining male mouse loses fecundity (Jarvis J.Eusociality in a mammal:cooperative
breeding in naked mole-rat colonies[J].Science.1981,4494(212):571-573.).Although
Male in naked mole group all possesses complete reproductive system structure, but the detection of serology hormonal readiness is found without life
Grow (Jarvis J, the Alexander R.The Biology of the Naked of testosterone etc. in the male naked mole body of ability
Mole-Rat [Z] .Princeton Univ, 1991275-336.) level is relatively low, and sperm quantity, form and locomitivity are notable
Less than that can educate individuality, this phenotype fits like a glove with the symptom of male sterility patient.Between the male of naked mole colony inside
The huge difference of naturally occurring fecundity becomes the ideal model of the research infertile disease of the mankind.But it is international at present
On research be only limitted to phenotype research, mechanism not yet low to testosterone levels in naked mole body launches research, biological among these
Once being elucidated with for mechanism, will have broad application prospects, especially to male sterility disease in clinical medicine domain
Prevention and treatment reference is great.
Original cuiture on interstitial cell is general all using (Klinefeher such as Klinefelter both at home and abroad at present
GR, Hall PF, Ewing LL.Effect of luteinizing hormone deprivation in situ on
steroidogenesis of rat Leydig cells purified by a muhistep procedure[J].Biol
Reprod, 1987,36 (3)：769-783.) method introduced, but because the method is harsh to experiment condition requirement, it is general real
Room is tested to be difficult to reach.And the extracorporeal culturing method research on naked mole interstitial glands is not more found at present.Mainly
Reason is：1) naked mole is a kind of poikilotherm, and the temperature of its body cell cultured in vitro needs to be groped, and its is non-constant
Organism metabolism speed causes its body cell in-vitro culture medium to be different from common rat or mouse；2) naked mole testis exists with epididymis
Functionally there is a certain degree of coincidence, testis goes back the place as storage sperm while sperm is produced, so being interstitial
The separation of cell brings certain difficulty；3) interstitial components ratio is high in naked mole testis, and interstitial cell separation method is different
In other rodents；4) naked mole has been already adapted to the environment of extraneous hypoxemia, so in its body cell cultured in vitro environment
Oxygen concentration be different from the animal lived in normal oxygen environment, and needs are groped.
This laboratory isolates and purifies naked mole testis by improvement using enzymic digestion and Percoll gradient of continuous density methods
Interstitial cell, and (Li is identified to resulting interstitial cell using anti-cholesterol side chain cleavage enzyme antibody (P450SCC)
Guangyu,Lan Haiyan,Liang Jihong,Mo yuncong,Deng Xuelian,Lin Chunyu,Su
Wenyong.2016.MCL1is a key regulator of steroidogenesis in mouse Leydig
cells.Mol Reprod Dev 83(3):226-35.), while the continuous monitoring ability of interstitial cell Testosterone Secretion.The party
The foundation of method will have laid a good foundation to study the function of interstitial cell from now on.
The content of the invention
It is an object of the invention to provide a kind of isolating and purifying and cultural method for naked mole interstitial glands, with side
Just the naked mole interstitial glands that purity is high, state is good, is efficiently obtained, is to set up naked mole interstitial glands in vitro to carry
For technical support, while for the function and biological characteristics of the naked mole interstitial glands of in vitro study are provided and establish theoretical base
Plinth.
To achieve these goals, the isolation and purification method of naked mole interstitial glands of the invention, including following step
Suddenly：
A, to adding digestive juice to be digested in naked mole testis tissue, after digestion terminates, add isometric digestion end
Only liquid terminates digestion；Postdigestive turbid solution (containing cell and tissue block) will be terminated to be filtered through 200 mesh cell screen clothes, to obtain
Single cell suspension；Then, cell suspension is placed in containing being centrifuged on 3 density gradient Percoll liquid levels；After centrifugation,
Visible 3 cell bands in pipe, collect the cell band between 35% and 60%, and fresh nutrient solution system is added after washing one time with PBS
Obtain cell suspension；
B, the suitable density of cell modulation is seeded in 60mm culture dishes, is cultivated in three gas incubators, cell
Most of adherent rear replacing nutrient solution；Culture carries out cellular identification after 7 days, centre changed a nutrient solution every 3 days.
Wherein, the naked mole testis tissue in the step A can be prepared by following steps：Animal dislocation is put to death
Afterwards, testis is taken out under aseptic condition, divests and an osculum is respectively cut into testis both sides after tunica albuginea.Testis is placed in aseptic PBS,
Intratesticular seminal fluid is tried one's best extrusion with tweezers, is so repeated 3 times.The testis tissue of removal sperm as far as possible is placed in fresh
In 1.8ml ice baths PBS, cut to 1mm with scissors3Size.
Further, the digestion condition in the step A is：Final concentration of 0.02mg/ml, DNA enzymatic I are dense eventually for type i collagen enzyme
It is 0.005% (mass volume ratio) to spend, and temperature is 32 DEG C, and the time is 30 minutes, and rotating speed is 30rpm/min.
Further, the digestion terminate liquid in the step A is to contain final concentration of 0.005% (mass volume ratio) DNA enzymatic I
Nutrient solution.
Further, the nutrient solution in the step A is：DMEM culture mediums+10% (V/V) hyclone+1% (V/V) is blue or green
Streptomysin+1% (V/V) glutamine.
Further, the Percoll concentration gradients in the step A are followed successively by, from top to bottom, respectively place 20%, 35%,
60% each 2.5ml.
Further, the centrifugal condition in the step A is 4 DEG C, 3000rpm centrifugations 30min.
Further, the cell density in the step B is 5-6 × 106Individual/ml.
Further, the parameter of three gas incubators in the step B is set to：At 34 DEG C, oxygen concentration is temperature control
10%, gas concentration lwevel is 5%, and humidity is 96%.
The beneficial effects of the present invention are：
1st, it is comprehensive to separate interstitial cell from naked mole testis tissue using various digestion separation methods, find out and be suitable to become
The reasonable cultural method of the naked mole interstitial glands of rodent mammal of temperature.We have found that under the conditions of 10% oxygen concentration,
Cell state can preferably be kept, and can keep increasing；
2nd, under the Percoll concentration gradients of 35%-60%, the naked mole interstitial glands of higher degree can be obtained.
In sum, the normal naked mole interstitial tissue[of testis] of a large amount of functional activities of acquisition that the inventive method can be easy, efficient, economic is thin
Culture under born of the same parents, hypoxia condition ensure that the biology that this cell can still be maintained under body state in ex vivo environment
Characteristic, consequently facilitating directly further studying the special of naked mole interstitial glands in pure in-vitro cell culture model
Physiological function, so as to provide important theoretical foundation to explore biological mechanism therein and being applied to clinical relevant.
Brief description of the drawings
Fig. 1 is the naked mole interstitial glands under ordinary optical microscope.
Fig. 2 is identified for the naked mole interstitial glands immunocytochemical stain of in vitro culture.Wherein A figures are
P450SCC antibody staining figures, B is Hoechst colored graphs, and C is two overlaps of figure of AB.
Specific embodiment
The specific embodiment that the present invention is provided is elaborated with reference to embodiment.
Embodiment 1
1st, experiment material
The naked mole of cleaning grade at 12 monthly ages is provided by Second Military Medical University, PLA's Experimental Animal Center.Will be naked
Mole is dislocated out after death, and skin degerming is carried out with 75% alcohol.Cut off from naked mole lower abdominal, taken from the place near dorsal part
Bilateral testes.
Type i collagen enzyme, DNA enzymatic I are purchased from Solarbio bio tech ltd, mycillin mixed liquor etc. and are purchased from
Sigma companies, DMEM, Australia source hyclone etc. are purchased from Thermo Fisher Scientific companies, 100 mesh cell sieves
Net purchase is purchased from Corning companies from BD companies, culture dish.
Interstitial glands medium component is DMEM+10% (V/V) hyclone+1% (V/V) paddy of mycillin+1%
Glutamine.
2nd, naked mole interstitial glands is isolated and purified and cultural method
Table 1
* P ＜ 0.05, compared with 0.002mg/ml groups；#P ＜ 0.05, ##P ＜ 0.01, compared with 0.02mg/ml groups.
As shown in the data of table 1, preferred type i collagen enzyme concentration is 0.02mg/ml.
Table 2
* P ＜ 0.05, compared with 5% oxygen concentration group；#P ＜ 0.05, compared with 10% oxygen concentration group.
As shown in the data of table 2, the preferred oxygen concentration of interstitial cell is 10%.
1. at animal dislocation after death, take out testis under aseptic condition, divest after tunica albuginea by testis both sides respectively cut one it is small
Mouthful.Testis is placed in aseptic PBS, intratesticular seminal fluid is tried one's best extrusion with tweezers, be so repeated 3 times.Essence will as far as possible be removed
The testis tissue of son is placed in fresh 1.8ml ice baths PBS, is cut to 1mm3 sizes with scissors.It is subsequently adding digestive juice (I type glue
Protoenzyme final concentration of 0.02mg/ml, DNA enzymatic I final concentration of 0.005% (mass volume ratio)), in 32 DEG C of constant temperature oscillation shaking tables
Digestion 30min, shaking speed is 30rpm/min.After digestion terminates, add isometric digestion terminate liquid (final concentration of
The nutrient solution of 0.005%DNA enzymes I) terminate digestion.Postdigestive turbid solution (containing cell and tissue block) will be terminated through 200 mesh
Cell screen clothes are filtered, to obtain single cell suspension.Then, cell suspension is placed in containing 3 density gradient Percoll (from upper
And under, 20%, 35%, 60% each 2.5ml is placed respectively) it is centrifuged on liquid level.After centrifugation, visible 3 cell bands in pipe,
The cell band between 35% and 60% is collected, is washed with PBS and is added fresh nutrient solution that cell suspension is obtained after one time.
2. by cell modulation 5-6 × 106It is seeded in after individual/ml in 60mm culture dishes, in three gas incubators, (temperature control exists
34 DEG C, oxygen concentration is 10%, and gas concentration lwevel is 5%, humidity be 96%) in cultivated, cell it is most of it is adherent after more
Change nutrient solution.Culture carries out cellular identification after 7 days, centre changed a nutrient solution every 3 days.
3rd, using the identification of the naked mole interstitial glands obtained by the present invention
The interstitial glands in proliferated culture medium is fixed using 4% paraformaldehyde, and combining form
The methods such as identification, immune refinement chemistry are identified.
1. cytomorphology identification：Result is as shown in figure 1, under light microscopic, interstitial glands cell space is in fusiformis or irregular
Ellipse, cell space diopter is stronger, there is the obvious halation of a circle around cell space, stretched out from cell space to surrounding two poles or three extremely dash forward
Rise.
2. immunocytochemistry identification：Result as shown in Fig. 2 using 4% paraformaldehyde will purify naked mole testis between
Cell plastid is fixed, and then carries out immunocytochemical stain using the antibody of specific anti-P450SCC.Result shows, cell energy
Enough keep PSC450SCC positive.
According to above-mentioned experimental result, the naked mole interstitial glands that the present invention is isolated and purified possesses typical interstitial cell
Form, it is positive in P450SCC, with bipolar or three pole projections, and good propagation shape can be kept in serum-containing media
State.
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described
Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (8)
1. a kind of isolation and purification method of naked mole interstitial glands, it is characterised in that comprise the following steps：
A, to adding digestive juice to be digested in naked mole testis tissue, after digestion terminates, add isometric digestion terminate liquid
Terminate digestion；The turbid solution containing cell and tissue block is filtered through 200 mesh cell screen clothes after digestion being terminated, unicellular to obtain
Suspension；Then, cell suspension is placed in containing being centrifuged on 3 density gradient Percoll liquid levels；After centrifugation, can in pipe
See 3 cell bands, collect the cell band between 35% and 60%, washed with PBS and add fresh nutrient solution that cell is obtained after one time
Suspension；
B, cell is seeded in 60mm culture dishes, is cultivated in three gas incubators, cell it is most of it is adherent after change training
Nutrient solution；Culture carries out cellular identification after 7 days, centre changed a nutrient solution every 3 days.
2. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Digestion condition in A is：Type i collagen enzyme final concentration of 0.02mg/ml, DNA enzymatic I final concentration of 0.005% (W/V), temperature is
32 DEG C, the time is 30 minutes, and rotating speed is 30rpm/min.
3. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Digestion terminate liquid in A is the nutrient solution containing final concentration of 0.005% (W/V) DNA enzymatic I.
4. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Nutrient solution in A is：DMEM culture mediums+10% (V/V) hyclone+1% (V/V) mycillin+1% (V/V) glutamine.
5. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Percoll concentration gradients in A are followed successively by, and from top to bottom, 20%, 35%, 60% each 2.5ml are placed respectively.
6. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Centrifugal condition in A is 4 DEG C, 3000rpm centrifugations 30min.
7. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
Cell density is 5-6 × 10 before inoculation in B6Individual/ml.
8. the isolation and purification method of naked mole interstitial glands according to claim 1, it is characterised in that the step
The parameter of three gas incubators in B is set to：At 34 DEG C, oxygen concentration is 10% to temperature control, and gas concentration lwevel is 5%, wet
Spend is 96%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192858A (en) * | 2018-01-12 | 2018-06-22 | 北京农学院 | A kind of method for preparing birds interstitial glands |
| CN109439615A (en) * | 2018-12-21 | 2019-03-08 | 贵州大学 | A method of the culture primary interstitial glands of high-purity Guizhou Xiang pig |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191124A (en) * | 2006-11-21 | 2008-06-04 | 中国人民解放军第二军医大学 | Separation method for Leydig cell and use thereof |
| WO2011061677A1 (en) * | 2009-11-17 | 2011-05-26 | Università degli Studi di Perugia | Method to prolong and improve the functionality of spermatozoa in vitro |
| CN104232575A (en) * | 2014-10-09 | 2014-12-24 | 广西大学 | Buffalo testicular interstitial cell isolated culture method |
-
2017
- 2017-01-06 CN CN201710009912.9A patent/CN106906177B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191124A (en) * | 2006-11-21 | 2008-06-04 | 中国人民解放军第二军医大学 | Separation method for Leydig cell and use thereof |
| WO2011061677A1 (en) * | 2009-11-17 | 2011-05-26 | Università degli Studi di Perugia | Method to prolong and improve the functionality of spermatozoa in vitro |
| CN104232575A (en) * | 2014-10-09 | 2014-12-24 | 广西大学 | Buffalo testicular interstitial cell isolated culture method |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192858A (en) * | 2018-01-12 | 2018-06-22 | 北京农学院 | A kind of method for preparing birds interstitial glands |
| CN108192858B (en) * | 2018-01-12 | 2020-07-07 | 北京农学院 | Method for preparing testis interstitial cells of poultry |
| CN109439615A (en) * | 2018-12-21 | 2019-03-08 | 贵州大学 | A method of the culture primary interstitial glands of high-purity Guizhou Xiang pig |
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