CN107858323B - Tortoise embryo fibroblast line and construction method thereof - Google Patents
Tortoise embryo fibroblast line and construction method thereof Download PDFInfo
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Abstract
The invention discloses a red-ear turtle embryo fibroblast line and a construction method thereof. Taking red-ear tortoise eggs incubated to 12-18 d, selecting live embryos, then sterilizing the red-ear tortoise eggs, removing shells to obtain the red-ear tortoise embryos, discarding heads, limbs and internal organs, and washing the remaining embryo tissues with PBS (phosphate buffered saline) containing double antibiotics until no obvious blood stains exist; digesting the embryo tissue which is washed to have no obvious blood stain by trypsin, collecting the digested single cells, inoculating the single cells into a complete culture medium for standing culture, sucking the complete culture medium and the cells which are not attached to the wall when the cells are partially attached to the wall and do not float by slight oscillation, and continuing to culture the remaining attached cells after adding a new complete culture medium; when the confluence degree of the primary cultured cells reaches more than 90%, the primary cultured cells are digested by trypsin, single cells are collected, and then inoculated in a complete culture medium for subculture. The invention lays a material and technical foundation for further utilizing RSTEFs to carry out basic theory and experimental research.
Description
The technical field is as follows:
the invention belongs to the technical field of cell culture, and particularly relates to a red-eared turtle embryo fibroblast line and a construction method thereof.
Background art:
red-eared turtle (trachelys script elegans), also known as brazilian turtle, is native to the middle of the united states to the north of mexico, and has been introduced into many countries in europe, africa, australia, asia, and america as a dangerous invasive species worldwide for international trade reasons. Meanwhile, the red-eared turtle is low in price and large in quantity, and is also an experimental animal for researches on zoology, physiology, medicine and the like. Researches show that the red-ear turtles have strong tolerance to temperature change, hunger, high salt stress and acid stress, however, at present, the researches mainly relate to the aspect of physiological and biochemical phenotypic traits and do not go deep into cytology and molecular mechanisms, and the important reason of the researches is probably that the in-vitro culture technology of the red-ear turtles is immature and the related cell lines capable of being used for experimental research are lacked. The only in vitro cultured turtle cells reported are cardiac cells of the carica carthami (terrene carpolina), liver and spleen cells of the western tortoise (chrysomys picta bellii), the snake alligator (Chelydra serpentina), the american turtle (Apalone microtia), 13 cells of the heart and liver of the green tortoise (Chelonia mydas), the red tortoise (cartacca), the olecranon (erette), the fibroblast of the tortoise (lepichthyophyta) and the cardiac cells of the chinese turtle. The method aims to establish a stable Red-ear slider turtle embryo fibroblast (RSTEFs) separation culture system, and lays a material and technical foundation for deeply researching the tolerance mechanism of the Red-ear turtle to adversity stress from the cell level and related physiology and medical research.
The invention content is as follows:
the invention aims to overcome the defects in the prior art, provides a red-eared turtle embryo fibroblast line and a construction method thereof, and lays a material and technical foundation for deeply researching the tolerance mechanism of the red-eared turtle to adversity stress from the cellular level and related physiological and medical researches.
The first purpose of the invention is to provide a method for constructing a red-ear turtle embryo fibroblast line, which comprises the following steps:
a) and (3) processing of embryos: taking the red-ear turtle eggs incubated to 12-18 d, selecting live embryos, then sterilizing the red-ear turtle eggs, removing shells to obtain the red-ear turtle embryos, removing heads, limbs and internal organs, and washing the remaining embryo tissues with PBS (phosphate buffered saline) containing double antibiotics until no obvious bloodstains exist;
b) primary culture: digesting the embryo tissue which is washed to have no obvious blood stain by trypsin, collecting the digested single cells, inoculating the single cells into a complete culture medium for standing culture, when the cells are partially attached to the wall and do not float after slight oscillation, sucking away the complete culture medium and the cells which are not attached to the wall, and continuing to culture the remaining attached cells after adding a new complete culture medium;
c) subculturing: when the confluence degree of the primary cultured cells reaches more than 90%, the primary cultured cells are digested by trypsin, single cells are collected, and then inoculated in a complete culture medium for subculture.
The method for sterilizing the eggs of the red-ear turtles after selecting the live embryos specifically comprises the following steps: soaking selected eggs of Testudinis with viable embryo in 6 vol% benzalkonium bromide solution for 3 min.
The inoculation ratio of the subculture is preferably 1: 2-3, or the inoculation density of the subculture is preferably 1.25-2.5 × 104/cm2。
The culture conditions of the primary culture and the subculture are preferably as follows: saturated humidity, 30-34 ℃ and 5% CO2. More preferably: saturated humidity, 34 ℃, 5% CO2。
The step of digesting the embryo tissues washed to have no obvious bloodstain by trypsin is specifically as follows: transferring the embryo tissue washed to have no obvious blood stain into a container containing 0.25% trypsin, repeatedly cutting into small pieces with ophthalmologic scissors, digesting in a water bath at 37 deg.C for 20min, periodically repeatedly blowing with a pipette, and adding complete culture medium to stop digestion.
The PBS containing the double antibody is preferably PBS containing 0.5kU/mL penicillin and 0.5mg/mL streptomycin.
The second purpose of the invention is to provide the red-ear turtle embryo fibroblast line constructed by the construction method of the red-ear turtle embryo fibroblast line.
According to the invention, a plasmid pDsRed2-N1 for coding red fluorescent protein and a plasmid pEGFP-C1 for coding green fluorescent protein are transfected into the red-ear turtle embryo fibroblast by using a liposome Lipofectamine 3000, and observation is carried out after 24h, 48h and 60h of transfection, so that only a small amount of cells can express the fluorescent protein at 24h, more fluorescent protein can be observed at 48h, fluorescent protein with higher density and brightness can be observed at 60h, the transfection rate is about 30%, and is obviously higher than the transfection rate of a Chinese softshell turtle heart cell line by 16%.
Therefore, the third objective of the present invention is to provide the application of the above-mentioned red-eared turtle embryo fibroblast cell line in cell transfection.
The invention successfully establishes a stable red-ear turtle embryo fibroblast line, and the obtained red-ear turtle embryo fibroblast (RSTEFs) has the characteristics of rapid growth, relatively low differentiation degree, relatively high in-vitro passage frequency and the like, and the RSTEFs can be used for cell transfection by a liposome-mediated method, the transfection rate is about 30 percent and is higher than the transfection rate of 16 percent of the cardiac cell line of the Chinese softshell turtle, thereby laying a foundation for further utilizing the RSTEFs to carry out basic theory and experimental research.
Description of the drawings:
FIG. 1 is a light mirror image of RSTEFs at different culture stages; wherein 1. primary RSTEFs are just inoculated into the light mirror image in the culture dish; 2. after standing still in the incubator for 30min, absorbing and removing the culture solution and the unadhered cells; 3, 4 and 5 are light microscopic images of RSTEFs after 24h culture, 72h culture and 12h first passage respectively; 6. mirror images of RSTEFs generation 5; mirror images of RSTEFs when they reach a certain density; 8. mirror images of RSTEFs generation 15; 9. mirror images of RSTEFs generation 6; the scale in the figure is 50 μm.
FIG. 2 is a diagram of liposome-mediated transfection of RSTEFs with pDsRed2-N1 and pEGFP-C1 plasmids; wherein 1, 2 and 3 are graphs of 60h rear light mirrors of plasmid pDsRed2-N1 transfected by RSTEFs; 4, 5 and 6 are graphs of 60h rear light mirrors after RSTEFs transfect pEGFP-C1 plasmid; the scale in the figure is 1000. mu.m.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
1 materials and methods
1.1 Red-ear turtle embryo Collection
Fertilized eggs of red-eared tortoise are collected from a breeding farm of the Guangdong mountain tortoise, the collected tortoise eggs are embedded into vermiculite (the mass ratio of vermiculite water is 1:0.8), and the fertilized eggs are transferred into a laboratory constant-temperature constant-humidity incubator for incubation within 24 hours after spawning, the incubation temperature is 32 ℃, and the humidity is 75-85%. And after incubation for 5-6 days, discarding the turtle eggs without obvious fertilized spots, and continuously incubating other fertilized eggs.
1.2 Biochemical reagents
Fetal bovine serum, 0.25% Trypsin (Trypsin), DMEM, Opti-MEM (from Gibco); DMSO, 100 XPicillin-streptomycin solution (penicillin 10kU/mL, streptomycin 10mg/mL), PBS, MTT cell proliferation assay kit (purchased from Biotechnology engineering, Inc.); lipofectamine 3000 (available from Thermo).
1.3 Experimental methods
RSTEFs separation and primary culture, selecting live embryo after hatching to 12d, soaking in 6% benzalkonium bromide solution for 3min, sterilizing egg shell, shelling, collecting embryo (which is an operation flow of embryo and can be used for operating multiple embryos at the same time), placing in a sterile flat dish, discarding head, limbs and internal organs, washing with PBS containing 5 × penicillin-streptomycin solution (i.e. PBS containing 0.5kU/mL penicillin and 0.5mg/mL streptomycin) for 3 times until no obvious blood stain exists, transferring into 2mL, adding 0.3mL 0.25% trypsin, repeatedly shearing into small pieces with ophthalmic scissors, placing in a 37 deg.C water bath for 20min, repeatedly blowing with 1mL liquid-moving gun every 5min during digestion, dispersing tissue pieces, adding 1mL complete culture medium to stop digestion when most tissue pieces are dispersed into single cell state, sucking, mixing, inoculating into 355 min, centrifuging, and inoculating with appropriate amount of 1.10% DMEM medium (containing 0.1-10% penicillin) to obtain supernatant, and centrifuging10cm cell culture dish, saturated humidity, 32 deg.C, 5% CO2And (3) standing and culturing for 30min in an incubator, observing the adherent condition of the cells under a mirror, sucking the culture solution together with the cells which are not attached to the wall into another culture dish when the cells are partially attached to the wall and do not float after slight oscillation, and continuing culturing after adding a new complete culture medium into the rest adherent fibroblasts.
Subculture of RSTEFs: taking the red-ear turtle embryo fibroblasts cultured in a 10cm cell culture dish out of an incubator, observing the cell density under a microscope, sucking out original culture solution when the confluence degree of the cells reaches 90%, washing the cells for 1 time by PBS (phosphate buffer solution), removing residual serum as much as possible, adding 1mL of 0.25% trypsin, gently shaking for several times to enable the trypsin to flow over the surfaces of all the cells, sucking out the digestive juice, digesting at room temperature for 3min, observing the digestion degree under the microscope, adding 5mL of complete culture medium to stop digestion when the adherent cells tend to be round and partially float, repeatedly and gently blowing the mixture by a pipette, transferring the mixture into a centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending the cells by 20mL of complete culture medium, and passaging the cells according to the ratio of 1: 2.
Cryopreservation of RSTEFs: preparing a proper amount of cell freezing medium (the cell freezing medium is DMEM medium containing 10% DMSO by volume fraction and 20% fetal calf serum by volume fraction), and precooling at 4 ℃. Taking cells growing to logarithmic phase, removing original culture solution by suction, and washing the cells for 1 time by using PBS (phosphate buffer solution), and removing residual serum as far as possible; digesting and collecting cells by the same method as subculture, sucking out cell sap after digestion is stopped, transferring the cell sap into a 15mL centrifuge tube, and centrifuging the cell sap for 5min at 1000 rpm; discarding the supernatant, adding appropriate amount of cell freezing solution to resuspend the cell precipitate, subpackaging into cell freezing tubes, placing into CoolCell programmed cooling box, freezing overnight at-80 deg.C, and storing in liquid nitrogen the next day for a long time.
Resuscitation of RSTEFs: firstly subpackaging 8mL of complete culture medium into 15mL centrifuge tubes, taking out the cryopreserved cells from a liquid nitrogen tank, clamping the tube opening with a hemostatic clamp, quickly placing the tube opening in a 37 ℃ water bath kettle, quickly shaking the cryopreserved tube to quickly thaw the tube, transferring the cryopreserved cells into a prepared 8mL complete culture medium to be gently mixed when the cells are just completely dissolved, and centrifuging the cell mixture at 1000rpm for 5minDiscarding supernatant, suspending cells with appropriate amount of fresh complete medium, transferring to cell culture dish, placing at saturated humidity, 32 deg.C, 5% CO2Culturing in an incubator.
MTT method detection cell growth curve: the 2 nd-generation RSTEFs were inoculated into 96-well plates at different concentrations, 100. mu.L of cell suspension per well, 4 multiple wells per group, and cultured continuously at 30 ℃, 32 ℃, 34 ℃ for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d, respectively. Then 10. mu.L of 5mg/mL MTT solution was added to each well, the culture medium in the wells was discarded after incubation in an incubator for 4 hours, 100. mu.L of DMSO solution was added, and the mixture was dissolved for 10min with shaking. The Optical Density (OD) of the cells was read at 540nm using a microplate reader. The time is used as an abscissa, and the OD value is used as an ordinate to draw an RSTEFs cell growth curve.
Cell transfection: when the degree of cell confluency was about 50%, the DNA-liposome complex was prepared in Opti-MEM serum-reduced medium according to the instructions of Lipofectamine 3000, and was directly added dropwise to the cultured cells uniformly, and the transfection results were observed at 24h, 48h, and 60h after transfection.
2 results
2.1 growth characteristics and morphological observations of RSTEFs
RSTEFs are inoculated into a culture dish for 30min to start to adhere to the wall, the wall is firmly adhered, and the RSTEFs cannot fall off even being slightly washed; after 72 hours of culture, the vast majority of adherent cells are in a typical fusiform shape, irregular star shape or polygonal shape, the cell volume is small, the number of mixed cells is small, the number of cytoplasmic granules is small, and the cytoplasm is uniform; the 2 nd-4 th generation cells have higher proliferation speed, are in the shape characteristics of typical fibroblasts, are consistent with the shape of primary cells, and gradually increase cells; the 5 th generation of cells has obviously increased volume and increased granules in cytoplasm; after passage for 5 times, cells which are flat, have obviously enlarged cytoplasmic vacuoles and enlarged intercellular spaces and are in an aging state can be observed; when the cells reach a certain density, the cells grow in a multilayer manner, are elongated in a fusiform manner, and are disorderly arranged; RSTEFs passed at least 15 generations, when cell morphology elongated significantly (FIG. 1).
When the cell confluence reaches 90%, digesting and freezing, preparing the cells into suspension by using a freezing solution, filling the suspension into a freezing tube, putting the freezing tube into a CoolCell program cooling box, directly placing the freezing tube into a refrigerator at minus 80 ℃, and transferring the freezing tube into liquid nitrogen for long-term storage the next day. When the cells are recovered, the frozen cells are quickly thawed in water bath at 37 ℃, the recovered cells can be quickly attached to the wall again after being inoculated to a culture dish, the growth speed and the form of the cells before and after recovery are not greatly different, and the cell survival rate is about 90%.
2.2 Effect of temperature on the growth of RSTEFs
RSTEFs by 2.5 × 104/cm2The density of (2) was inoculated in a 96-well culture plate, and the plate was continuously cultured at 30 ℃ and 32 ℃ and 34 ℃ for 8 days, each group was provided with 4 multiple wells, and OD of cells cultured in 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d was measured by MTT method540The value is obtained. OD of 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d cultured cells at 34 ℃540The values are respectively 0.456 +/-0.013, 0.590 +/-0.011, 0.774 +/-0.040, 0.880 +/-0.028, 1.006 +/-0.030, 1.133 +/-0.043, 1.286 +/-0.047 and 1.271 +/-0.038; OD of cells cultured at 32 ℃ for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d540The values are respectively 0.468 +/-0.004, 0.559 +/-0.007, 0.701 +/-0.007, 0.776 +/-0.022, 0.850 +/-0.006, 0.905 +/-0.028, 1.052 +/-0.013 and 1.095 +/-0.017; OD of 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d cultured cells at 30 ℃540The values are 0.484 + -0.009, 0.580 + -0.012, 0.650 + -0.010, 0.735 + -0.016, 0.765 + -0.011, 0.864 + -0.007, 0.919 + -0.017 and 0.973 + -0.010, respectively. The RSTEFs have good growth state under the conditions of 30 ℃, 32 ℃ and 34 ℃, can enter an exponential growth phase, and the growth speed of the RSTEFs is increased along with the increase of the temperature.
2.3 Effect of seeding Density on the growth of RSTEFs
RSTEFs were each assigned 2.5 × 104/cm2、1.25×104/cm2、6.25×103/cm2、3.125×103/cm2Inoculating four density gradients in 96-well culture plate, setting 4 multiple wells in each group, continuously culturing in 34 deg.C incubator for 8d, and detecting OD of cells cultured in 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d by MTT method540Value. inoculum density was 2.5 × 104/cm2OD of cells cultured for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d540The values are 0.364 + -0.002, 0.484 + -0.009, 0.580 + -0.012, 0.650 + -0.010,0.735 + -0.016, 0.765 + -0.011, 0.864 + -0.007 and 0.919 + -0.017, and the inoculation density is 1.25 × 104/cm2OD of cells cultured for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d540The values are respectively 0.227 plus or minus 0.006, 0.250 plus or minus 0.026, 0.312 plus or minus 0.011, 0.414 plus or minus 0.004, 0.489 plus or minus 0.014, 0.541 plus or minus 0.015, 0.642 plus or minus 0.022 and 0.714 plus or minus 0.008, and the inoculation density is 6.25 × 103/cm2OD of cells cultured for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d540The values are respectively 0.173 + -0.004, 0.196 + -0.008, 0.222 + -0.022, 0.240 + -0.007, 0.268 + -0.006, 0.277 + -0.011, 0.328 + -0.013 and 0.363 + -0.005, the inoculation density is 3.125 × 103/cm2OD of cells cultured for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d540The values are respectively 0.141 + -0.006, 0.156 + -0.005, 0.156 + -0.004, 0.166 + -0.003, 0.171 + -0.006, 0.170 + -0.003, 0.183 + -0.002 and 0.210 + -0.005, the inoculation density is 2.5 × 104/cm2And 1.25 × 104/cm2RSTEFs under the condition has good growth state, can smoothly enter logarithmic growth phase, and has the inoculation density of 6.25 × 103/cm2、3.125×103/cm2RSTEFs grow slowly and are difficult to proliferate under the conditions, and the results show that the inoculation density of the RSTEFs is not lower than 1.25 × 104/cm2。
2.4 transfection of RSTEFs
The plasmid pDsRed2-N1 coding the red fluorescent protein and the plasmid pEGFP-C1 coding the green fluorescent protein are transfected into RSTEFs by using a liposome Lipofectamine 3000, and observation is carried out after 24h, 48h and 60h of transfection, only a small number of cells can express the fluorescent protein in 24h, more fluorescent protein can be observed in 48h, fluorescent protein with higher density and brightness can be observed in 60h, and the transfection efficiency is about 30% (figure 2). The morphology of the cells after transfection is different from that before transfection, and the cells have the tendency of elongation and thinning.
Example 2:
1 materials and methods
1.1 Red-ear turtle embryo Collection
Fertilized eggs of red-eared tortoise are collected from a breeding farm of the Guangdong mountain tortoise, the collected tortoise eggs are embedded into vermiculite (the mass ratio of vermiculite water is 1:0.8), and the fertilized eggs are transferred into a laboratory constant-temperature constant-humidity incubator for incubation within 24 hours after spawning, the incubation temperature is 32 ℃, and the humidity is 75-85%. And after incubation for 5-6 days, discarding the turtle eggs without obvious fertilized spots, and continuously incubating other fertilized eggs.
1.2 Biochemical reagents
Fetal bovine serum, 0.25% Trypsin (Trypsin), DMEM, Opti-MEM (from Gibco); DMSO, 100 XPicillin-streptomycin solution (penicillin 10kU/mL, streptomycin 10mg/mL), PBS, MTT cell proliferation assay kit (purchased from Biotechnology engineering, Inc.); lipofectamine 3000 (available from Thermo).
1.3 Experimental methods
RSTEFs separation and primary culture, selecting live embryo after hatching to 18d, irradiating egg with fiber-optic cold light source, soaking for 3min with 6% benzalkonium bromide solution, sterilizing egg shell, shelling, collecting embryo of Chinemys reevesii (which is an operation procedure of embryo and can operate multiple embryos at the same time), placing into a sterile flat dish, discarding head, limbs and viscera, washing for 3 times with PBS containing 5 × penicillin-streptomycin solution (i.e. PBS containing 0.5kU/mL penicillin and 0.5mg/mL streptomycin), transferring into 2mL centrifuge tube, adding 0.5mL 0.25% trypsin, repeatedly shearing into small pieces with ophthalmic scissors, placing into a 37 deg.C water bath for 20min, repeatedly blowing with 1mL liquid-moving gun every 5min, dispersing tissue pieces, when most tissue pieces are dispersed into a state, adding 1mL complete culture medium, stopping digestion, sucking, mixing, inoculating into 1000 mL serum, centrifuging, adding appropriate amount of 0.5 rpm, inoculating to 3510% saturated culture medium containing 10% penicillin, and adding DMEM, and culturing2And (3) standing and culturing for 30min in an incubator, observing the adherent condition of the cells under a mirror, sucking the culture solution together with the cells which are not attached to the wall into another culture dish when the cells are partially attached to the wall and do not float after slight oscillation, and continuing culturing after adding a new complete culture medium into the rest adherent fibroblasts.
Subculture of RSTEFs: taking the red-ear turtle embryo fibroblasts cultured in a 10cm cell culture dish out of an incubator, observing the cell density under a microscope, sucking out original culture solution when the confluence degree of the cells reaches 90%, washing the cells for 1 time by PBS (phosphate buffer solution), removing residual serum as much as possible, adding 1mL of 0.25% trypsin, gently shaking for several times to enable the trypsin to flow over the surfaces of all the cells, sucking out the digestive juice, digesting for 5min at room temperature, observing the digestion degree under the microscope, adding 5mL of complete culture medium to stop digestion when the adherent cells tend to be round and partially float, repeatedly and gently blowing the mixture by a pipette, transferring the mixture into a centrifuge tube, centrifuging for 5min at 1000rpm, discarding supernatant, resuspending the cells by 30mL of complete culture medium, and carrying out cell passage according to the proportion of 1: 3.
Cryopreservation of RSTEFs: preparing a proper amount of cell freezing medium (the cell freezing medium is DMEM medium containing 10% DMSO by volume fraction and 20% fetal calf serum by volume fraction), and precooling at 4 ℃. Taking cells growing to logarithmic phase, removing original culture solution by suction, and washing the cells for 1 time by using PBS (phosphate buffer solution), and removing residual serum as far as possible; digesting and collecting cells by the same method as subculture, sucking out cell sap after digestion is stopped, transferring the cell sap into a 15mL centrifuge tube, and centrifuging the cell sap for 5min at 1000 rpm; discarding the supernatant, adding appropriate amount of cell freezing solution to resuspend the cell precipitate, subpackaging into cell freezing tubes, placing into CoolCell programmed cooling box, freezing overnight at-80 deg.C, and storing in liquid nitrogen the next day for a long time.
Resuscitation of RSTEFs: firstly, subpackaging 8mL of complete culture medium based on 15mL centrifuge tubes, taking out the cryopreserved cells from a liquid nitrogen tank, clamping the tube orifice with a hemostatic clamp, quickly placing the tube into a 37 ℃ water bath kettle, quickly shaking the cryopreserved tube to quickly thaw the tube, transferring the cryopreserved cells into 8mL of complete culture medium prepared in advance when the cells are just completely dissolved, gently mixing the cells uniformly, centrifuging the cells for 5min at 1000rpm, discarding supernatant, suspending the cells with a proper amount of fresh complete culture medium, transferring the cells into a cell culture dish, placing the cell culture dish in saturated humidity, 32 ℃ and 5% CO2Culturing in an incubator.
MTT method detection cell growth curve: the 2 nd-generation RSTEFs were inoculated into 96-well plates at different concentrations, 100. mu.L of cell suspension per well, 4 multiple wells per group, and cultured continuously at 30 ℃, 32 ℃, 34 ℃ for 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d, respectively. Then 10. mu.L of 5mg/mL MTT solution was added to each well, the culture medium in the wells was discarded after incubation in an incubator for 4 hours, 100. mu.L of DMSO solution was added, and the mixture was dissolved for 10min with shaking. The Optical Density (OD) of the cells was read at 540nm using a microplate reader. The time is used as an abscissa, and the OD value is used as an ordinate to draw an RSTEFs cell growth curve.
Cell transfection: when the degree of cell confluency was about 50%, the DNA-liposome complex was prepared in Opti-MEM serum-reduced medium according to the instructions of Lipofectamine 3000, and was directly added dropwise to the cultured cells uniformly, and the transfection results were observed at 24h, 48h, and 60h after transfection.
2 results
2.1 growth characteristics and morphological observations of RSTEFs
RSTEFs are inoculated into a culture dish for 30min to start to adhere to the wall, the wall is firmly adhered, and the RSTEFs cannot fall off even being slightly washed; after 72 hours of culture, the vast majority of adherent cells are in a typical fusiform shape, irregular star shape or polygonal shape, the cell volume is small, the number of mixed cells is small, the number of cytoplasmic granules is small, and the cytoplasm is uniform; the 2 nd-4 th generation cells have higher proliferation speed, are in the shape characteristics of typical fibroblasts, are consistent with the shape of primary cells, and gradually increase cells; the 5 th generation of cells has obviously increased volume and increased granules in cytoplasm; after passage for 5 times, cells which are flat, have obviously enlarged cytoplasmic vacuoles and enlarged intercellular spaces and are in an aging state can be observed; when the cells reach a certain density, the cells grow in a multilayer manner, are elongated in a fusiform manner, and are disorderly arranged; RSTEFs can reach at least 15 generations, when the cell morphology is obviously elongated.
When the cell confluence reaches 90%, digesting and freezing, preparing the cells into suspension by using a freezing solution, filling the suspension into a freezing tube, putting the freezing tube into a CoolCell program cooling box, directly placing the freezing tube into a refrigerator at minus 80 ℃, and transferring the freezing tube into liquid nitrogen for long-term storage the next day. When the cells are recovered, the frozen cells are quickly thawed in water bath at 37 ℃, the recovered cells can be quickly attached to the wall again after being inoculated to a culture dish, the growth speed and the form of the cells before and after recovery are not greatly different, and the cell survival rate is about 90%.
2.2 Effect of temperature on the growth of RSTEFs
RSTEFs by 2.5 ×104/cm2The cell density of (2) is inoculated in a 96-well culture plate, the culture plate is respectively placed at 30 ℃, 32 ℃ and 34 ℃ for continuous culture for 8 days, each group is provided with 4 multiple wells, and a cell growth curve under different temperatures is drawn by utilizing an MTT method. The RSTEFs have good growth state under the conditions of 30 ℃, 32 ℃ and 34 ℃, can enter an exponential growth phase, and the growth speed of the RSTEFs is increased along with the increase of the temperature.
2.3 Effect of seeding Density on the growth of RSTEFs
RSTEFs were each assigned 2.5 × 104/cm2、1.25×104/cm2、6.25×103/cm2、3.125×103/cm2Inoculating four density gradients in 96-well culture plate, setting 4 multiple wells in each group, continuously culturing in 34 deg.C incubator for 8 days, and drawing cell growth curve by MTT method under different inoculation density conditions, wherein the inoculation density is 2.5 × 104/cm2And 1.25 × 104/cm2RSTEFs under the condition has good growth state, can smoothly enter logarithmic growth phase, and has the inoculation density of 6.25 × 103/cm2、3.125×103/cm2RSTEFs grow slowly and are difficult to proliferate under the conditions, and the results show that the inoculation density of the RSTEFs is not lower than 1.25 × 104/cm2。
2.4 transfection of RSTEFs
The plasmid pDsRed2-N1 for coding red fluorescent protein and the plasmid pEGFP-C1 for coding green fluorescent protein are transfected to RSTEFs by using a liposome Lipofectamine 3000, and observation is carried out after 24h, 48h and 60h of transfection, only a small number of cells can express the fluorescent protein in 24h, more fluorescent protein can be observed in 48h, fluorescent protein with higher density and brightness can be observed in 60h, and the transfection efficiency is about 30%. The morphology of the cells after transfection is different from that before transfection, and the cells have the tendency of elongation and thinning.
The invention adopts the red-ear tortoise embryos incubated at 32 ℃ to 12-18 d as the source of RSTEFs, the length of the red-ear tortoise embryos at the stage is 1-2 cm, the heads, the tails, the limbs and the internal organs are clear and can be distinguished and easily peeled off, and 3-6 × 10 can be obtained from each embryo6The cell amount of the primary cells is large and the purity is high. If the embryo age is too short, the embryo is very youngSmall and too soft, the operation is very inconvenient, and the head, the tail, the limbs and all internal organs of the fetus are difficult to completely remove, so that the obtained fibroblasts are easy to be mixed with mixed cells, thereby affecting the quality of the cultured fibroblasts. However, the embryo with too long age has differentiated the dorsal concha, so the embryo is not easy to be stripped and the connection between cells is difficult to digest.
It is critical that the primary cell culture prevent cell contamination. During sampling, the red-ear tortoise egg is first soaked in benzalkonium bromide solution for disinfection to avoid surface contamination, and the embryo separating process is preferably performed in a clean bench while the time of exposing the embryo in air is minimized. The invention adopts the ophthalmic scissors to firstly cut the tissues in a centrifuge tube, then the tissues are digested by pancreatin, and the cells are purified by utilizing the fact that the adherence speed of fibroblasts is higher than that of other cells. The whole process is quick and simple, the probability of cell pollution is small, and the success rate is high.
The red-ear turtle is a temperature-variable animal, has a relatively wide tolerance range to temperature, and has a certain elastic range compared with mammals, and the requirement on temperature for cell culture is not high. The result of the invention shows that RSTEFs can normally grow and proliferate at 30-34 ℃, and the growth rate is highest at 34 ℃. The inoculation density has great influence on the growth of RSTEFs, cells with lower inoculation density are more prone to aging, and the RSTEFs can directly fail to passage due to the excessively low inoculation density.
For limited cells, genetic traits are subject to change after multiple passages in vitro culture. Mouse embryonic fibroblasts generally grow only well for 5 passages, and cells die rapidly after 5 passages. After 10 generations, rabbit embryonic fibroblasts showed signs of aging, with flat cell bodies, increased pseudopodia and slow growth rate. The experimental results show that RSTEFs cells before the 5 th generation grow rapidly, are vigorous in metabolism and regular in cell shape, and the aging phenomenon appears after the 5 th generation of partial cell strains. Most cell lines can reach at least 15 generations, but the cell morphology is different from that of the primary cell, and the cell is obviously elongated.
The invention successfully establishes the red-ear turtle embryo fibroblast line, the obtained RSTEFs have the characteristics of fast growth, relatively low differentiation degree, relatively high in-vitro passage frequency and the like, the RSTEFs can be used for cell transfection by a liposome-mediated method, the transfection rate is about 30 percent and is higher than the transfection rate of 16 percent of the cardiac cell line of the Chinese softshell turtle, and a material and technical basis is laid for further utilizing the RSTEFs to carry out basic theory and experimental research.
Claims (9)
1. A method for constructing an Erythrocea sinensis embryo fibroblast line is characterized by comprising the following steps:
a) and (3) processing of embryos: taking the red-ear turtle eggs incubated to 12-18 d, selecting live embryos, then sterilizing the red-ear turtle eggs, removing shells to obtain the red-ear turtle embryos, removing heads, limbs and internal organs, and washing the remaining embryo tissues with PBS (phosphate buffered saline) containing double antibiotics until no obvious bloodstains exist;
b) primary culture: digesting the embryo tissue which is washed to have no obvious blood stain by trypsin, collecting the digested single cells, inoculating the single cells into a complete culture medium for standing culture, when the cells are partially attached to the wall and do not float after slight oscillation, sucking away the complete culture medium and the cells which are not attached to the wall, and continuing to culture the remaining attached cells after adding a new complete culture medium;
c) subculturing: when the confluence degree of the primary cultured cells reaches more than 90%, digesting the primary cultured cells by trypsin, collecting single cells, and then inoculating the single cells into a complete culture medium for subculture;
the inoculation density of subculture is 1.25-2.5 × 104/cm2。
2. The method for constructing a tortoise embryo fibroblast line of the red fungus according to claim 1, wherein the step of sterilizing the tortoise eggs after selecting the viable embryos comprises: soaking selected eggs of Testudinis with viable embryo in 6 vol% benzalkonium bromide solution for 3 min.
3. The method for constructing the red-ear turtle embryo fibroblast line according to claim 1, wherein the inoculation ratio of subculture is 1: 2-3.
4. The method for constructing a red-eared turtle embryo fibroblast line according to claim 1, wherein the culture conditions of the primary culture and the subculture are as follows: saturated humidity, 30-34 ℃ and 5% CO2。
5. The method for constructing a red-ear turtle embryo fibroblast cell line according to claim 4, wherein the culture conditions of the primary culture and the subculture are as follows: saturated humidity, 34 ℃, 5% CO2。
6. The method for constructing a red-ear turtle embryo fibroblast cell line according to claim 1, wherein the trypsinizing the embryo tissue washed to no obvious blood stain comprises: transferring the embryo tissue washed to have no obvious blood stain into a container containing 0.25% trypsin, repeatedly cutting into small pieces with ophthalmologic scissors, digesting in a water bath at 37 deg.C for 20min, periodically repeatedly blowing with a pipette, and adding complete culture medium to stop digestion.
7. The method for constructing a red-ear turtle embryo fibroblast cell line of claim 1, wherein the PBS containing double antibody is PBS containing 0.5kU/mL penicillin and 0.5mg/mL streptomycin.
8. The red-eared turtle embryo fibroblast line constructed by the method for constructing a red-eared turtle embryo fibroblast line according to any one of claims 1 to 7.
9. Use of the red-eared turtle embryonic fibroblast cell line of claim 8 in cell transfection.
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