CN101942499A

CN101942499A - Method for detecting cell apoptosis in germ cell-supporting cell coculture

Info

Publication number: CN101942499A
Application number: CN2009100546876A
Authority: CN
Inventors: 吴庆; 赖纯米; 李克勇; 常秀丽; 周志俊
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2009-07-10
Filing date: 2009-07-10
Publication date: 2011-01-12

Abstract

The invention belongs to the technical field of biology, and relates to a method for detecting cell apoptosis in germ cell-supporting cell coculture. In the method, the cell apoptosis in the coculture is detected by directly performing TUNEL straining and contamination on germ cells and supporting cells cocultured in a short term in a culture dish. Through the method, a germ cell-supporting cell coculture form can be kept and the most typical biochemical characteristics and morphological characteristics of the cell apoptosis are accurately reflected as the TUNEL staining is directly performed in the culture dish; and simultaneously, the success rate of an experiment is increased as the processes of coverglass smearing and the like which require corresponding technology are not required. The method can be used for observing the acute reproductive toxicity and the cell apoptosis of chemicals by utilizing a germ cell-supporting cell coculture system, and is proved by the experiment to be credible.

Description

A kind of method that detects sexual cell-sustenticular cell co-cultured cell apoptosis

Technical field

The invention belongs to biological technical field, relate to and detect apoptotic method, be specifically related to a kind of sexual cell-sustenticular cell cultured cells apoptosis detection method altogether.

Background technology

It is owing to simulated physiological disposition that testis sexual cell and sustenticular cell are cultivated altogether, and can improve the survival rate of sexual cell and promote the proliferate of sexual cell, is usually used in sexual cell grown the research of atomization and cultivate sexual cell being used for transplanting.At present, the genotoxicity research that utilizes this cultivation system to inquire into chemicals still is in the initial stage.

When inquiring into chemicals toxicity, the influence of chemicals pair cell apoptosis is a common counter.Prior art detects in the toxic method of chemicals, although wherein the chromosome fluorescence staining is easy, sample should not be preserved; Breach end-labelling (TUNEL) with the mediation of deoxyribonucleotide terminal enzyme (DNA), as long as breach occurs on dna double splitting of chain or the chain, be labeled under the effect of transferring enzyme (TdT) endways, it is highly sensitive, not needing large-scale instrument, is to survey apoptotic common method at present.

Prior art is to culturing cell during with the TUNEL method, and cell is handled nothing more than being divided into two kinds of situations, and the one, attached cell is taked cell climbing sheet, the 2nd, suspension cell is taked smear behind the collecting cell.But, research report is arranged, when observing rat testicle sexual cell-sustenticular cell and cultivate the apoptosis of system altogether,, brought difficulty for general observational technique because existing adherent sustenticular cell has the sexual cell of suspension again in the co-culture system.Simultaneously owing to cultivate altogether that sexual cell and sustenticular cell coexist mutually in the system, collecting cell and creep plate smear can not be observed its whole apoptosis situation.Still the apoptotic report during discovery is cultivated altogether with TUNEL method detection rat testicle sexual cell-sustenticular cell at present.For this reason, the present invention intends exploring Short-term Culture, directly in culture dish co-cultured cell system is carried out the painted method of TUNEL.

Summary of the invention

The purpose that the present invention is is to overcome the deficiencies in the prior art, provides a kind of detection apoptotic method, is specifically related to a kind of sexual cell-sustenticular cell cultured cells apoptosis detection method altogether.

The present invention's employing in culture dish short-term is cultivated sexual cell altogether and sustenticular cell carries out direct TUNEL dyeing, detects whole cell apoptosis situation in the cultivation altogether.

Particularly, sexual cell-sustenticular cell provided by the invention is cultured cells apoptosis detection method altogether, it is characterized in that separating male testis sexual cell of rat and sustenticular cell by former generation, in culture dish, sexual cell and sustenticular cell are cultivated altogether, contamination, directly observe the apoptosis situation of cultivating system altogether with the breach end-labelling (TUNEL) of deoxyribonucleotide terminal enzyme (DNA) mediation in culture dish, it comprises the steps:

(1) rat male sex-cell, the former generation separation of sustenticular cell are cultivated in the culture dish

(2) apoptosis dyeing and observation

Among the present invention, described step 1), laboratory animal sexual cell, sustenticular cell separation and Culture are:

Get newborn male rat, sterilize after the conventional processing, get the both sides testis under the aseptic condition fast, place the capsule that fills HBSS, HBSS cleans the back and moves into centrifuge tube, adds the DMEM liquid effect 20min that contains 100 μ g/mLDNAase, 0.1% collagenase and 0.1% Unidasa; The centrifugal supernatant that goes, add the DMEM liquid effect 30min that contains 100 μ g/mLDNAase, 0.1% collagenase again, the centrifugal supernatant that goes, add DMEM liquid and place sucking-off supernatant behind the ice bath, add pancreatin piping and druming, microscopically is observed and is added 2%FBS termination digestion, the centrifugal supernatant that goes after seminiferous tubule becomes one cell, add fresh medium, make single cell suspension and be used for cell cultures; In the culture dish with cell inoculation gelatin paving, place 5%CO2,95% air saturation humidity incubator to cultivate.

Among the present invention, described step 2) apoptosis dyeing and being viewed as:

Cell is handled: in culture dish, need not to take out, by fix time cultivate after, the sucking-off nutrient solution, with 4% Paraformaldehyde 96 fixed cell, fixing 1h; According to operation shown in the TUNEL test kit (commercial).

Experimental result shows

1. TUNEL staining cell apoptosis is consistent with expection under different culture temperature and different incubation times.

Relatively cultivate 2h for 33 ℃, 41 ℃ of cultivation 2h and 41 ℃ of apoptosis situations of cultivating 14h, result show that 41 ℃ of incidences of cultivating 2h group and 14h group gained apoptosis are all apparently higher than 33 ℃ of cultivation 2h groups, through χ ²Check its difference that statistical significance is arranged.

2. the performance of TUNEL staining cell apoptosis is consistent with expection under different concns cadmium exposure condition.

Cultivating is after cultivation made sexual cell adherent fully in 24 hours, to add different concns cadmium (0,2.5,5,10 μ m) and cultivate 12 hours observation of cell apoptosis.The result shows, except sexual cell and sustenticular cell apoptosis image, still has sustenticular cell to engulf apoptotic cell image (see figure 3).

It is acute genotoxicity and the apoptosis of observing chemicals that the present invention utilizes sexual cell-sustenticular cell to cultivate altogether, can overcome the defective that the caused cellular form of collecting cell changes in ordinary method, can keep the state of sexual cell-when the sustenticular cell different shape is cultivated altogether to carry out TUNEL dyeing, thus most typical biological chemistry of reacting cells apoptosis and morphological specificity accurately; Do not need simultaneously cell climbing sheet smear etc. to need the process of relevant art, improved the success ratio of experiment.

Description of drawings

Fig. 1 is the sustenticular cell and the sexual cell growing state of common cultivation

Show that wherein cultivate 2h, sustenticular cell begins adherent, sexual cell is suspension; Cultivate 14h, visible most sustenticular cells are adherent, and sexual cell is attached at sustenticular cell.(

Fig. 2 be different culture temperature with different incubation times under apoptosis figure shown in the TUNEL dyeing.

Fig. 3 cultivates the 24 hours apoptosis figures ' of contamination cadmium after 12 hours

Wherein show, except sexual cell and sustenticular cell apoptosis image, still have sustenticular cell to engulf the apoptotic cell image.

Embodiment

Embodiment 1

1. laboratory animal sexual cell, sustenticular cell separation and Culture

Get and give birth to the newborn male rat in back, 75% ethanol disinfection after the conventional processing, get the both sides testis under the aseptic condition fast, place the capsule that fills HBSS, careful down fatty tissue, epididymis, testis tunicle and the blood vessel of removing each testis of anatomical lens, HBSS cleans the centrifuge tube that the back moves into 15ml, adds the DMEM liquid effect 20min that contains 100 μ g/mLDNAase, 0.1% collagenase and 0.1% Unidasa that is equivalent to 10 times of tissue volume.The centrifugal supernatant that goes, add the DMEM liquid effect 30min that contains 100 μ g/mLDNAase, 0.1% collagenase again, the centrifugal supernatant that goes, add DMEM liquid and place sucking-off supernatant behind the ice bath 15min, add pancreatin piping and druming, observe seminiferous tubule to microscopically and become to add 2%FBS behind the one cell and stop digestion, the centrifugal supernatant that goes, (DMEM+ Sodium.alpha.-ketopropionate+non-essential amino acid+Sodium.alpha.-hydroxypropionate+microbiotic+Transferrins,iron complexes+LIF) make single cell suspension is used for cell cultures to add the 2ml fresh medium.In the culture dish of using the gelatin paving, cell inoculation density is 70-80% with cell inoculation, places 5%CO2,95% air saturation humidity incubator to cultivate.

2. apoptosis dyeing and observation

Cell is handled: in culture dish, need not to take out, by fix time cultivate after, the sucking-off nutrient solution, with 4% Paraformaldehyde 96 fixed cell, fixing 1h.Shown in the TUNEL test kit, inhale and remove stationary liquid, add PBS and wash 2 times; Add 3%H2O incubated at room 10min; Wash 2 times with PBS; Add the mark damping fluid (the reagent negative control adds the reaction solution that does not contain the TdT enzyme) contain TdT and DIG-d-UTP, culture dish is placed wet box, in 37 ℃ of marks 2 hours; TBS liquid adds confining liquid after washing 2 times, incubated at room 30min; Inhale deblocking liquid, do not wash and add 1: 100 biotinylation anti digoxin antibody diluent 50 μ L, culture dish is placed wet box, 37 ℃ of reaction 30min in marked region; Add 1 after washing 2 times with TBS: 100SABC antibody diluent 50 μ L place wet box in marked region with culture dish, 37 ℃ of reaction 30min; Wash 4 times with TBS; Add the liquid that DAB colour developing liquid develops the color to culture dish and become brown (about 15min), the distillation washing is not inhaled, and inverted microscope is observation down.

Experimental result shows

1, TUNEL staining cell apoptosis is consistent with expection under different culture temperature and different incubation times.

Sustenticular cell, spermatogonium are cultivated altogether 33 ℃ of cultivation 2h apoptosis incidences and are no more than 2% according to the literature, and obvious apoptosis will take place 41 ℃ of cultivations, therefore the present invention has compared 33 ℃ of cultivation 2h, cultivates 2h and 41 ℃ of apoptosis situations of cultivating 14h for 41 ℃.The result shows that 41 ℃ of incidences of cultivating 2h group and 14h group gained apoptosis are all apparently higher than 33 ℃ of cultivation 2h groups, through χ ²Check its difference that statistical significance is arranged.

Table 1 selects 3 visuals field (every group of visual field of all selecting same position) for single blind method and counts apoptotic cell and total cell count (at least 100 cells in each visual field) gained apoptosis rate in each visual field in the painted zone of mark.

Table 1

2, the performance of TUNEL staining cell apoptosis is consistent with expection under different concns cadmium exposure condition.

Table 2 is that cadmium contamination back apoptosis increases, and is dose-dependence, through its difference of χ2Jian Yan statistical significance is arranged.

Table 2

+: be that 2.5 μ M group compares with 0 μ M group

#: be that 2.5 μ M group compares with 5.0 μ M group

*: be that 5.0 μ M group compares with 10.0 μ M group

Claims

1. one kind is detected the method that chemicals are induced sexual cell-sustenticular cell co-cultured cell apoptosis, it is characterized in that separating male testis sexual cell of rat and sustenticular cell by former generation, in culture dish, sexual cell and sustenticular cell are cultivated altogether, contamination is directly observed the apoptosis situation of cultivating system altogether with the breach end-labelling of deoxyribonucleotide terminal enzyme (DNA) mediation in culture dish; It comprises the steps:

(1) laboratory animal sexual cell, the former generation separation of sustenticular cell are cultivated in the culture dish;

(2) apoptosis dyeing and observation.

2. by the described method of claim 1, it is characterized in that, in the described step (1), 75% ethanol disinfection behind the conventional processing laboratory animal male rat, get the both sides testis under the aseptic condition fast, place the capsule that fills HBSS, HBSS cleans the centrifuge tube that the back moves into 15ml, adds the DMEM liquid effect 20min that contains 100 μ g/mLDNAase, 0.1% collagenase and 0.1% Unidasa; The centrifugal supernatant that goes, add the DMEM liquid 30min that contains 100 μ g/mLDNAase, 0.1% collagenase again, the centrifugal supernatant that goes, add DMEM liquid and place sucking-off supernatant behind the ice bath 15min, add pancreatin piping and druming, add 2%FBS termination digestion after microscopically observation seminiferous tubule becomes one cell, the centrifugal supernatant that goes adds fresh medium 2ml and makes single cell suspension; Inoculating cell places 5%CO2,95% air saturation humidity incubator to cultivate in the culture dish in gelatin paving.

3. by the described method of claim 2, it is characterized in that in the described step (1), described fresh medium contains DMEM+ Sodium.alpha.-ketopropionate+non-essential amino acid+Sodium.alpha.-hydroxypropionate+microbiotic+Transferrins,iron complexes+LIF.

4. by the described method of claim 2, it is characterized in that in the described step (1), described cell inoculation density is 70-80%.

5. by the described method of claim 1, it is characterized in that in the described step (2), cultivating altogether is that the sucking-off nutrient solution is with 4% Paraformaldehyde 96 fixed cell 1h after cultivating; Stationary liquid is removed in suction, adds PBS and washes 2 times; Add 3%H2O incubated at room 10min; Wash 2 times with PBS; Add the mark damping fluid that contains TdT and DIG-d-UTP; Culture dish is placed wet box, 37 ℃ of marks 2 hours; TBS liquid adds confining liquid after washing 2 times, incubated at room 30min; Inhale deblocking liquid, do not wash and add 1: 100 biotinylation anti digoxin antibody diluent 50 μ L, culture dish is placed wet box, 37 ℃ of reaction 30min in marked region; Add 1 after washing 2 times with TBS: 100SABC antibody diluent 50 μ L place wet box in marked region with culture dish, 37 ℃ of reaction 30min; Wash 4 times with TBS; It is brown adding DAB colour developing liquid to liquid, and observations under the inverted microscope is not inhaled in the distillation washing.