Application of Smad6 gene in promoting reprogramming of fibroblasts into stem cells

Abstract

The present invention discloses the application of Smad6 gene in promoting fibroblast reprogramming into stem cells. The application is specifically to inhibit Smad2 / 3 activation by overexpressing Smad6 gene, promote the upregulation of C‑Myc, Nanog and Oct4, important transcription factors for maintaining and regulating stem cell state, thereby promoting fibroblast reprogramming into stem cells. The present invention uses fish oocyte extract as an inducer to induce fibroblast reprogramming into stem cells, and the materials are cheap, extensive and safe. The present invention promotes the reprogramming of fibroblasts induced by fish oocyte extract into stem cells by overexpressing Smad6 gene, promotes the expression of stem cell related genes C‑Myc, Nanog and Oct4, inhibits the activation of TGF‑β signaling pathway, regulates the expression of TGF‑β signaling pathway related genes Smad2, Smad3 and Smad4, improves the efficiency of somatic cell reprogramming into stem cells, and greatly promotes the application of reprogramming.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of Smad6 gene in promoting the reprogramming of fibroblasts into stem cells. Background Art

[0002] Reprogramming technology can transform adult cells into cells with stem cell properties, which also changes the ethical issues caused by the use of human embryonic stem cells. At the same time, the emergence of this technology has also shown great application prospects in regenerative medicine research, disease treatment research and drug screening. So far, a variety of disease-specific reprogrammed cell lines have been successfully established and used to explore therapeutic effects, mainly including Alzheimer's disease, spinal muscular atrophy, Parkinson's disease, Duchenne muscular dystrophy, diabetes, liver disease, β-thalassemia, etc. The results show that inducing reprogrammed cells can significantly alleviate the clinical symptoms of these diseases.

[0003] The development of reprogramming technology has overcome the defects of scarce embryonic stem cell sources and large individual differences, and avoided a series of ethical controversies caused by the destruction of embryos. Therefore, it has broader application prospects in disease simulation, drug screening, personalized treatment, and research on the mechanism of early embryonic development, and has become a hot topic in the field of stem cell research. Over the past half century, researchers have tried a variety of technical means to reprogram somatic cells and have made a series of breakthroughs. With the development of reprogramming technology, there are now many reprogramming methods that can be used, including transcription factor-mediated reprogramming, small molecule compound-mediated reprogramming, cell fusion technology, epigenetic regulation, gene editing technology, etc.

[0004] In recent years, a method for reprogramming using oocytes has also been developed. N Kikyo et al. used extracts from African clawed frog oocytes to treat the epithelial cells of clawed frogs and found that histones in the epithelial cells of clawed frogs changed. Currently, there are also many reprogramming studies using this method. In 2011, Wu Sujun et al. used bovine oocytes to induce bovine fibroblasts to reprogram and detected Nanog and Oct-4Gene expression. In 2013, Fan Zongxing used the oocyte extract of African clawed frogs to conduct a reprogramming experiment on bovine somatic cells. In 2015, Du Weihua and others found that the oocyte extract of clawed frogs could be used to induce partial reprogramming of bovine fetal fibroblasts, which could restore the developmental totipotency of bovine fetal fibroblasts. Based on the experience of using oocytes of African clawed frogs, we also realized whether we could use the fish oocyte extract, which is cheap and widely available, for induction. In this way, the experimental materials are cheaper, more widespread, and non-toxic. Based on previous research, we have also obtained some remarkable results, such as the method of preparing fish oocyte extract, the optimal concentration for inducing mouse skin fibroblasts to reprogram, the changes in the morphology and proliferation rate of fibroblasts after induction, the successful induction of reprogrammed cells to differentiate into other types of cells, and the main role of proteins in fish oocyte extracts.

[0005] The emergence of reprogramming is not only conducive to the research of anti-aging mechanisms and drugs, but also conducive to the formation of new cell therapies to treat body injuries and diseases. At present, some products related to fish oocytes have been applied. In terms of diet, eating caviar (usually refers to the delicacy made from sturgeon eggs) helps to maintain skin health, promote skin cell regeneration, reduce wrinkles and improve skin tone, fight aging and certain diseases; in beauty and health care, in some high-end products, caviar peptides extracted from fish roe are extracted and used as active ingredients, which can promote skin repair and regeneration, reduce fine lines and wrinkles, and increase skin elasticity and radiance.

[0006] At present, this reprogramming technology still has many problems. This is mainly reflected in the following aspects: First, the stability of the active ingredients in the fish oocyte extract is poor. This active ingredient cannot exist in the fish oocyte extract for a long time. After a period of storage, the effect of inducing reprogramming will decrease. Secondly, the use of fish oocyte extract to induce mouse skin fibroblast reprogramming cannot guarantee the reprogramming efficiency.

[0007] The present invention aims to provide a method for improving the efficiency of reprogramming fish oocyte-induced fibroblasts into stem cells. Summary of the invention

[0008] The purpose of the present invention is to provide an application of the Smad6 gene in promoting the reprogramming of fibroblasts into stem cells. The application is specifically to inhibit the activation of Smad2 / 3 by overexpressing the Smad6 gene, promote the upregulation of important transcription factors C-Myc, Nanog and Oct4 that maintain and regulate the stem cell state, thereby promoting the reprogramming of fibroblasts into stem cells.

[0009] The method for promoting the reprogramming of fibroblasts into stem cells by Smad6 gene is achieved by the following steps: transfecting fibroblasts with a lentiviral vector overexpressing SMAD6, and then using fish oocyte extract to induce the reprogramming of the transfected fibroblasts into stem cells.

[0010] The beneficial effects of the present invention are as follows: the present invention uses fish oocyte extract as an inducer to induce fibroblast reprogramming into stem cells, and the material is cheap, extensive and safe. The present invention promotes the reprogramming of fibroblasts induced by fish oocyte extract into stem cells by overexpressing the Smad6 gene, promotes the expression of stem cell-related genes C-Myc, Nanog and Oct4, inhibits the activation of the TGF-β signaling pathway, regulates the expression of TGF-β signaling pathway-related genes Smad2, Smad3 and Smad4, improves the efficiency of somatic cell reprogramming into stem cells, and greatly promotes the application of reprogramming. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a flow chart of the detection of the regulatory effect of plv-Smad6 on reprogramming in Example 16;

[0012] Figure 2 The groups for the pre-overexpression experiment in Example 1 were as follows (Note: Group M: complete culture medium, to observe the infection effect of the virus on cells under conventional culture conditions; Group A: complete culture medium + HiTransG A group, to observe whether HiTransG A can improve the infection effect; Group P: complete culture medium + HiTransGP group, to observe whether HiTransG P can improve the infection effect; Control group: to monitor whether the cell growth is normal during the experiment).

[0013] Figure 3 The situation of plv-Smad6-HSF at different MOIs in Example 1 (4×);

[0014] Figure 4 The figure is the Smad6 lentivirus infection figure of Example 1 (10×), wherein A: plv-Smad6-HSF figure; B: mRNA expression level of plv-Smad6-HSF; C: relative mRNA expression level of C-Myc, Nanog, and Oct4 genes in plv-Smad6-HSF (Note: ns represents P﹥0.05, *** represents P﹤0.001, and **** represents P﹤0.0001);

[0015] Figure 5 The sample loading position of the precast gel in Example 1;

[0016] FIG6 shows the relative expression levels of mRNA of each gene in plv-Smad6 of Example 2 (Note: ns represents P>0.05, **** represents P<0.0001);

[0017] Figure 7 Figure A in the middle shows the protein expression of each gene in the TGF-β signaling pathway in plv-Smad6, and Figure B shows the statistical analysis of the protein expression of each gene in the TGF-β signaling pathway (Note: ** represents P﹤0.01, **** represents P﹤0.0001);

[0018] Figure 8 This is a map of sample loading positions for precast gels;

[0019] Fig. 9 This is the result of Coomassie Brilliant Blue staining. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0021] The present invention provides an application of the Smad6 gene in promoting the reprogramming of fibroblasts into stem cells. The application specifically inhibits the activation of Smad2 / 3 by overexpressing the Smad6 gene, promotes the upregulation of important transcription factors C-Myc, Nanog and Oct4 that maintain and regulate the stem cell state, and thus promotes the reprogramming of fibroblasts into stem cells.

[0022] The method for promoting the reprogramming of fibroblasts into stem cells by Smad6 gene is achieved by the following steps: transfecting fibroblasts with a lentiviral vector overexpressing SMAD6, and then using fish oocyte extract to induce the reprogramming of the transfected fibroblasts into stem cells.

[0023] Example 1 Preparation of inducer fish oocyte extract

[0024] The live crucian carp purchased from Zhuanxin Market in Kunming was selected in this experiment. The crucian carp with oocyte yolk filled should be selected. The preparation method of fish oocyte extract was obtained according to the summary of the previous experiment.

[0025] (1) Remove the roe from the fish belly on ice, then grind the roe in a pre-cooled mortar until the roe becomes a paste with no obvious roe cells.

[0026] (2) Transfer the ground fish eggs to a 50 ml centrifuge tube and add 0.9% NaCl solution at a volume ratio of 1:1. Use a vortex oscillator to oscillate and mix until the ground fish eggs are evenly distributed in the 0.9% NaCl solution.

[0027] (3) Centrifuge in a high-speed refrigerated centrifuge at 4°C, 3000 rpm for 10 min.

[0028] (4) Take the supernatant and place it in a new 50 ml centrifuge tube. Continue to place it in a desktop high-speed refrigerated centrifuge and centrifuge it at 4°C, 12,000 rpm for 10 min. Take the supernatant.

[0029] (5) Use a 0.45µm Millex GP disposable syringe filter and a 0.22µm Millex GP disposable syringe filter to filter and sterilize the supernatant to obtain the fish oocyte extract. Place the filtered and sterilized supernatant in a 4°C refrigerator for short-term storage.

[0030] Example 2

[0031] The fibroblasts were reprogrammed into stem cells by overexpressing the Smad6 gene. The experimental process is as follows Figure 1 The specific experimental steps are as follows:

[0032] 1. Construction of Smad6 gene lentiviral overexpression vector and virus embedding (this process was performed by Shanghai Genetech Technology Co., Ltd.)

[0033] (1) Design a lentiviral expression vector that contains a lentiviral packaging signal, a replication origin, a selection marker, and a multiple cloning site.

[0034] (2) Obtain the Smad6 gene by PCR amplification or cloning method and insert it into the multiple cloning site of the lentiviral vector.

[0035] (3) Verify the correctness of the vector construction, usually through restriction analysis and DNA sequencing.

[0036] (4) Transfect the constructed Smad6 gene lentiviral vector into the packaging cell line.

[0037] (5) After transfection, cells begin to produce lentiviral particles carrying the Smad6 gene, which are released from the packaging cells.

[0038] (6) Collect and concentrate the released lentiviral particles carrying the Smad6 gene for use in subsequent cell transfection experiments.

[0039] In this experiment

[0040] 2. Cell transfection and transfection efficiency detection

[0041] The fibroblasts after digestion were counted using a cell counting plate. According to the counting results, complete culture medium (i.e., DMEM / F12 culture medium containing 10% fetal bovine serum) was used to obtain 2 ml of 3×10 4 100µl of the prepared cell suspension was added to 16 wells of the 96-well plate. Four wells were selected as the control group. The other 12 wells were set up as three experimental groups (Group M: complete culture medium, to observe the infection effect of the virus on cells under normal culture conditions; Group A: complete culture medium + virus infection enhancement solution A, to observe whether virus infection enhancement solution A can improve the infection effect; Group P: complete culture medium + virus infection enhancement solution P, to observe whether virus infection enhancement solution P can improve the infection effect), with four wells in each group. The experimental layout of the 96-well plate is as follows Figure 2 Then, the 96-well plate was placed in a 37°C CO2 incubator for 16-24 hours until the cell confluence was observed to be 20-30% under a microscope. Then, the lentivirus carrying the Smad6 gene was taken out of the -80°C ultra-low temperature refrigerator and placed on an ice box to slowly melt the lentivirus.

[0042] The lentivirus carrying the Smad6 gene was diluted to a titer of 1×10 8 TU / ml, 5×10 7 TU / ml, 2×10 7 TU / ml, 1×10 7 TU / ml, each titer is required to reach at least 35µl to provide sufficient virus volume for subsequent experiments.

[0043] Discard the original cell culture medium in each well of the 96-well plate, add complete culture medium, lentivirus carrying Smad6 gene and virus infection enhancement solution according to Table 1, and use a 100µl pipette to slowly blow and mix the mixed solution in each well. Then spray alcohol on the surface of the 96-well plate, wipe it dry and place it in a 37℃ CO2 incubator for continued culture. After 8-16 hours of culture, take the 96-well plate out of the CO2 incubator. According to the cell growth state observed under the microscope, when the cell morphology changes, the medium can be replaced with DMEM / F12 culture medium containing 10% fetal bovine serum, spray alcohol on the surface, wipe it dry and place it in a 37℃ CO2 incubator for continued culture. After 24-48 hours of culture, it can be observed under the microscope that fibroblasts have high fluorescence expression. Generally, after 72 hours of infection, fibroblasts will have a relatively high infection rate of about 80%. The results at this time can be used as the infection conditions of fibroblasts and the selected MOI in subsequent experiments.

[0044] Table 1 Pre-infection experimental conditions

[0045]

[0046] 3. Puromycin sensitivity test

[0047] To the fibroblasts with a confluence of 80%, 0µl, 1µl, 2µl, 3µl, 4µl, 5µl, 6µl, 7µl, 8µl, 9µl, 10µl of 2mg / ml Puromycin were added respectively. The fibroblasts were cultured in the culture medium containing 0µg / ml, 1µg / ml, 2µg / ml, 3µg / ml, 4µg / ml, 5µg / ml, 6µg / ml, 7µg / ml, 8µg / ml, 9µg / ml, 10µg / ml Puromycin respectively. After culturing in a 37℃ CO2 incubator for 24-48h, the death of fibroblasts was observed, and the optimal concentration of Puromycin that caused fibroblast death was screened out, paving the way for subsequent experimental studies.

[0048] 4. Detection of plv-Smad6 cell phenotype and stem cell marker expression

[0049] The fibroblasts after digestion were counted using a cell counting plate, and a concentration of 3×10 was obtained using DMEM / F12 medium containing 10% fetal bovine serum according to the counting results. 4 / ml cell suspension. Add 2ml of cell suspension to each well of the 6-well plate, spray alcohol on the surface and wipe it dry, then place it in a 37℃ CO2 incubator for 16-24h. Under a microscope, it can be observed that the cell fusion of fibroblasts reaches 20-30%. At this time, take out the lentivirus carrying the Smad6 gene prepared in step 1 from the -80℃ ultra-low temperature refrigerator and put it on an ice box to slowly melt the lentivirus carrying the Smad6 gene. Use DMEM / F12 medium containing 10% fetal bovine serum to dilute the virus to a titer of 5×10 7TU / ml. Remove the original cell culture medium from each well, select three wells in the 6-well plate, add 50µl of lentivirus carrying the Smad6 gene, 40µl of virus infection enhancement solution P infection solution, and 910µl of DMEM / F12 medium containing 10% fetal bovine serum to each well. Add 1mL of DMEM / F12 medium containing 10% fetal bovine serum to the other three wells as the control group. Use a 1000µl pipette to mix. Spray alcohol on the surface of the 6-well plate, wipe it dry and place it in a 37℃ CO2 incubator for continued culture. After 8-16h of culture, take the 6-well plate out of the CO2 incubator. According to the cell growth state observed under the microscope, when the cell morphology changes, the medium can be replaced with DMEM / F12 medium containing 10% fetal bovine serum, and then spray alcohol on the surface, wipe it dry and place it in a 37℃ CO2 incubator for continued culture. About 72h after infection, observe the infection efficiency of lentivirus carrying the Smad6 gene on fibroblasts under a microscope.

[0050] Remove the original cell culture medium from each well of the 6-well plate, add 1ml of DMEM / F12 containing 10% fetal bovine serum and 3µl of 2mg / ml puromycin, spray the surface with alcohol, wipe it dry and place it in a 37℃ CO2 incubator for culture. After 24-48h, it was observed under a microscope that the unsuccessfully infected fibroblasts were almost completely dead, and then the cell RNA and protein were extracted for later experimental detection. After cell counting, 6000 cells / cm 2 Inoculate the cells into the cell culture flask at a seeding density of 100 μg / cm2 and shake gently to make them uniform; after standing, spray alcohol on the surface of the cell culture flask, wipe it dry and gently place it in a 37°C incubator containing 5% CO2. After culturing for 24-48 hours, replace the cell culture medium when the cell fusion degree reaches 80% under a microscope. Add an appropriate amount of fish oocyte extract to the cell culture flask to induce fibroblast reprogramming. The final concentration of fish oocyte extract is

[0051] 10µg / ml. After gently shaking, spray alcohol on the surface of the cell culture bottle, wipe it dry and gently place it in a 37℃ 5% CO2 incubator for 24 hours before proceeding to the next experimental operation.

[0052] 5. Extract RNA

[0053] (1) Transfer the reprogrammed cells into a 1.5 ml EP tube, add 1 ml of Trizol reagent, vortex and mix well, and place on ice for 5 min;

[0054] (2) Add 1 / 5 of the amount of chloroform to the tube in a ratio of 5:1, cap the tube tightly, vortex and mix for 15 seconds, then leave it at room temperature for 3 minutes to allow for natural phase separation;

[0055] (3) Place the EP tube in a desktop high-speed refrigerated centrifuge and centrifuge at 4°C, 10,000 g for 15 min. After centrifugation, the mixture will separate into three layers: the upper layer is a colorless aqueous phase, the middle layer is white, and the lower layer is a red phenol-chloroform organic phase. The RNA to be extracted is mainly concentrated in the aqueous phase.

[0056] (4) Use a 1000µl pipette to transfer 400-500µl of the supernatant from the centrifuged mixture into a new EP tube. Add an equal volume of chloroform to the supernatant to the EP tube, vortex and mix for 15 seconds, then place at room temperature for 10 minutes to allow for natural phase separation.

[0057] (5) Centrifuge at 12,300 rcf for 10 min at 4°C and discard the supernatant.

[0058] (6) Add pre-cooled 75% ethanol to the precipitate to wash the RNA precipitate, vortex and mix well, centrifuge at 7700 rcf at 4°C for 5 min, and discard the supernatant.

[0059] (7) Repeat step (6) and carefully remove excess liquid with a pipette tip;

[0060] (8) Place the RNA precipitate on a clean bench for 10 minutes to air dry, and dissolve the RNA precipitate with an appropriate amount of enzyme-free water.

[0061] 6. RNA concentration detection

[0062] (1) Turn on the instrument and click Nucleic Acid Determination on the main interface;

[0063] (2) Use an alcohol cotton ball to clean the sample window on the base and the mirror in the cover arm, and then wipe it again with a dry cotton ball;

[0064] (3) Use an integrated vortex mixer to mix the test sample;

[0065] (4) Lift the cover arm and use a pipette to add 1 µl of saline solution to the sample injection port on the base;

[0066] (5) Lower the cover arm and start the blank test;

[0067] (6) Clean the measuring window and mirror on the cover arm with a wet alcohol cotton ball, and then wipe it again with a dry cotton ball;

[0068] (7) Lift the cover arm and add 1 µl of sample solution to the sample port.

[0069] (8) After the measurement is completed, lift the cover arm, use a moistened alcohol cotton ball and a dry cotton ball to clean the surface and test the next sample.

[0070] 7. Reverse transcription

[0071] (1) According to the RNA concentration obtained by the test, use the formula (the amount of RNA template added = 500 ng / the RNA concentration obtained by the test) to calculate the amount of RNA template to be added, and then put the extracted RNA and various reagents in the kit into an ice box for melting and use;

[0072] (2) According to Table 2, add each component of the reaction system (10 µl) into a 200 µl enzyme-free eight-tube strip on ice;

[0073] Table 2 Reverse transcription 10µl system

[0074]

[0075] (3) Use a vortex oscillator to oscillate the reaction system, then place it in a low-speed centrifuge for a short centrifugation. Next, place it in a PCR instrument and set it to 42°C for 2 min and 60°C for 5 min for incubation.

[0076] (4) After the incubation is completed, remove the eight-tube strip from the PCR instrument and quickly place it on an ice box to cool it down. Next, place it in a low-speed centrifuge for a short centrifugation and use a pipette to add the components in Table 3 in sequence.

[0077] Table 3 Reverse transcription 20µl system

[0078]

[0079] (5) Use a vortex oscillator to mix the reaction system, and then place it in a low-speed centrifuge for a short centrifugation. Next, place it in a PCR instrument and set it to 25℃ for 10 min, 55℃ for 15 min, and 85℃ for 5 min for incubation.

[0080] (6) After the incubation is completed, remove the eight-tube strip from the PCR instrument, quickly place it on an ice box to cool it, and refrigerate it for later use.

[0081] 8. RT-qPCR semi-quantitative detection

[0082] According to Table 4, add each component of the RT-qPCR reaction system (20 µl) into 100 µl eight-tube strips in sequence on an ice box;

[0083] The reaction system was mixed by vortex oscillator, then placed in a low-speed centrifuge for brief centrifugation, placed in a quantitative PCR instrument, and the reaction program was set: 95°C pre-denaturation for 1 min, 1 cycle; cyclic reaction (95°C 10s-60°C 30s), 45 cycles. The upstream and downstream primer sequence information of the gene used is shown in SEQ ID NO: 1-10.

[0084] Table 4 RT-qPCR reaction system

[0085]

[0086] Table 5 Primer sequences of Smad6 gene

[0087]

[0088] result:

[0089] As shown in Figure 3, there was no or little fluorescence at MOI=10 and MOI=20, indicating that the efficiency of lentiviral transfection was not high; at MOI=50, the efficiency of lentiviral transfection in group P was better; at MOI=100, the efficiency of lentiviral transfection in groups M, A and P was very high, but the fluorescence was too dense, making it difficult to further analyze the images later.

[0090] Fibroblasts were transfected with lentivirus containing Smad6 for 16 hours, and then the medium was changed and cultured for another 72 hours before observation under a fluorescence microscope. Figure 4 It can be seen that the cell transfection coverage rate of the overexpression group is above 80%. Compared with the normal cultured fibroblasts, the relative expression level of Smad6 gene in plv-Smad6 is as follows Figure 4 .Depend on Figure 4 As shown in A, the relative expression of Smad6 gene increased significantly, and there was a significant difference compared with the control group, indicating that the effect of overexpression of Smad6 gene was extremely significant. In addition, the relative expression levels of C-Myc, Nanog and Oct4 were significantly upregulated, suggesting that overexpression of Smad6 gene can change the expression levels of stem cell markers Nanog and Oct4.

[0091] In summary, overexpression of Smad6 can promote the reprogramming of fibroblasts into stem cells.

[0092] Example 3 Effect of Smad6 gene on TGF-β signaling pathway

[0093] 1. The specific operation method is the same as step 8 of Example 1. Among them, Smad2, Smad3, ID2, ID3, Smad1, Smad5

[0094] The primer base sequences of the Smad8 and Smad8 genes are shown in Table 6 , and the primer base sequences of the GAPDH gene are the same as those in Table 5 .

[0095] Table 6 Primer sequences of Smad6 gene

[0096]

[0097] 2. Western blot protein detection

[0098] (1) Protein extraction: Pour out the original culture medium in the 6-well plate, wash it 2-3 times with pre-cooled PBS, and then use a pipette to clean the remaining PBS in the 6-well plate. Prepare an ice box, prepare lysis solution at a ratio of 1ml RIPA +10µl PMSF, add 60-80µl lysis solution to each well of the 6-well plate, place it on ice for 20 minutes, and scrape the cells 3-4 times during this period to allow the cells and lysis solution to fully react. Then transfer all the solutions in the 6-well plate to an EP tube, place it in a desktop high-speed refrigerated centrifuge and centrifuge it at 4℃ and 12000rpm for 10 minutes. Then carefully remove the EP tube after centrifugation, and transfer the supernatant of the solution to a new EP tube.

[0099] (2) Protein concentration determination:

[0100] 1) Add the standard volume and standard dilution into a 96-well plate according to Table 7.

[0101] Table 7 Standard volume, amount of standard diluent added and final concentration

[0102]

[0103] 2) Add 20 μl of fish oocyte extract or 20 μl of fish oocyte extract diluted a certain multiple into the wells of a 96-well plate;

[0104] 3) Add 200 μl of BCA working solution to each well for fish oocyte extract detection and protein concentration standard curve preparation, seal the 96-well plate with sealing film, and then place it in a 37°C electric constant temperature drying oven for 30 minutes;

[0105] 4) Use a microplate reader to select the absorbance of the wavelength between A540-595nm to measure the fish oocyte extract and the standard;

[0106] 5) Draw a standard curve y=kx+b based on the absorbance of the standard measured by the microplate reader;

[0107] 6) Substitute the measured absorbance of the fish oocyte extract into the equation to obtain the X value in the equation, which is the protein concentration in the fish oocyte extract. (If the added fish oocyte extract is diluted, it needs to be further multiplied by the dilution multiple).

[0108] (3) Preparation of loading protein

[0109] 1) Add the components in Table 7 into the EP tube in sequence:

[0110] Table 7 Preparation system of sample protein

[0111]

[0112] Use a vortex oscillator to oscillate the reaction system, then place it in a low-speed centrifuge for a short centrifugation, insert it into a foam plate, and then boil it in boiling water for 10 minutes. After cooling the cooked protein on ice, it can be directly used for subsequent Western blot experiments or stored in a -80℃ refrigerator.

[0113] 2) SDS-PAGE gel electrophoresis: The specific operation method is the same as step 2.1.4 (2). Figure 5 shown.

[0114] 3) Transfer: According to the size of the gel required later, choose the size of the PVDF membrane and then cut it. Soak the cut PVDF membrane in methanol for about 1 minute. Referring to the marker bands running on the gel, cut out the approximate area where the target protein is located, and soak it in the transfer solution. Make a "sandwich" (negative electrode-black filter-three-layer filter paper-cut precast gel-PVDF membrane-three-layer filter paper-black filter-positive electrode). During the production process, each layer needs to be continuously poured with transfer solution and bubbles must be removed. After the "sandwich" is made, close and clamp the clamps, then put it in the transfer tank and add enough transfer solution. Put ice packs in and around the transfer box, turn on the power, set 260m for transfer for 1h;

[0115] 4) Blocking of membrane: After the transfer, remove the PVDF membrane from the "sandwich" and place it in the 5% skim milk prepared in advance, and block it on a shaker at room temperature for more than 2 hours;

[0116] 5) Incubation with primary antibody: After blocking, wash the PVDF membrane on a shaker with TBST solution, repeat 3 times, 10 minutes each time; place the PVDF membrane in the diluted primary antibody and incubate at 4°C overnight;

[0117] 6) Incubation with secondary antibody: On the second day, wash the PVDF membrane on a shaker with TBST solution, repeat 3 times, 10 min each time; place the washed PVDF membrane in the diluted secondary antibody and incubate on a shaker at room temperature in the dark for 1 hour; wash the PVDF membrane on a shaker with TBST solution, repeat 3 times, 15 min each time;

[0118] 7) ECL color development: Prepare ECL working solution (solution A: solution B = 1:1) (keep away from light); use a pipette to add the working solution to the surface of the PVDF membrane so that the part of the PVDF membrane with antibodies is completely covered. Then, place it in a gel imager for development and save the image. Use image J software to analyze the captured image.

[0119] result:

[0120] 1. mRNA level expression detection

[0121] As shown in Figure 6A, the relative expression levels of Smad2 and Smad3 genes in the TGF-β signaling pathway and the relative expression levels of ID2 and ID3 genes located downstream of the TGF-β signaling pathway have decreased significantly, which is consistent with the data results of the transcriptome obtained in the previous study. At the same time, the relative expression levels of the BMP signaling pathway (Smad5 and Smad8 genes) associated with the TGF-β signaling pathway have also been downregulated, as shown in Figure 6A. Figure 6 B.

[0122] 2. Protein expression level detection

[0123] Western Blot experiments were performed to detect the changes in genes in the TGF-β signaling pathway in the control group fibroblasts and plv-Smad6. The results are shown in Figure 7 It can be seen that compared with fibroblasts without overexpression experiments, the expression levels of Smad2 and Smad3 gene proteins in the overexpressed TGF-β signaling pathway, activated P-Smad2 and P-Smad3 proteins, combined Smad2 / Smad3 proteins, and Smad4 protein combined with the Smad2 / 3 complex in the downstream of the pathway were significantly reduced.

[0124] Experimental Example 1 Detection of relative molecular weight of fish oocyte extract protein prepared in Example 1

[0125] (1) Cooking egg whites

[0126] Determine the protein concentration of the fish oocyte extract. According to the measured protein concentration of the fish oocyte extract, add 5× loading buffer and PBS solution to make the final protein concentration of the fish oocyte extract between 10-30 mg / ml. Use a vortex oscillator to oscillate and mix, and put it into boiling water for denaturation in a boiling water bath for 15 minutes. The denatured protein is stored in a -80°C refrigerator.

[0127] (2) SDS-PAGE electrophoresis

[0128] 1) Glue loading: Open two 10% precast gels, place the glass side of the precast gel facing you, and insert it into the electrophoresis tank. Add a concave slot outside the precast gel plate, and place the gel in the middle (with the comb inside). After both precast gels are loaded into the electrophoresis tank, pour the electrophoresis solution into the inner tank to fill it up and check for leaks. After soaking for 3 minutes, use both hands to evenly force the comb upwards and take it out steadily. Put the inner tank into the electrophoresis box, and continue to add electrophoresis solution to the inner tank until it reaches 1 / 3 of the height of the outer tank.

[0129] 2) Sample loading: Add 20µl sample protein and 5µl marker to the sample well of the precast gel, and add 5µl of 5× loading buffer on both sides, as shown in Figure 8. Cover the electrophoresis box, turn on the power, set the constant voltage to 80V and start electrophoresis. After about 20 minutes, when the front of the bromophenol blue dye electrophoreses to the junction of the two gels, immediately change to 115V constant voltage for electrophoresis; after 1 hour of electrophoresis, wait until the front of the bromophenol blue dye electrophoreses to the end and turn off the power.

[0130] (3) Washing gel

[0131] Place the precast gel into the modified pipette box, add 50 ml of sterile water, heat in a 70°C electric constant temperature drying oven for 8 minutes, place on a shaker and shake horizontally for 5 minutes, carefully pour out the sterile water in the box, and repeat the washing operation 3 times to remove impurities such as SDS in the gel.

[0132] (4) Dyeing

[0133] Remove the sterile water remaining in the pipette box, add about 50 ml of Coomassie Brilliant Blue Rapid Staining Solution to stain the gel, and place it on a shaker at room temperature for 30 minutes. Pour out the Coomassie Brilliant Blue Rapid Staining Solution in the gun box and remove the remaining Coomassie Brilliant Blue Rapid Staining Solution with a pipette.

[0134] (5) Decolorization

[0135] Add about 50 ml of Coomassie Brilliant Blue staining destaining solution to the gun box, making sure that the destaining solution can fully cover the gel. Then put a piece of absorbent paper in to absorb the destaining dye, and then place it on a shaker and shake it slowly horizontally. Destain for 6 hours at room temperature. During this period, replace the Coomassie Brilliant Blue staining destaining solution in the gun box every 2 hours.

[0136] (6) Photo recording and preservation

[0137] After the decolorization is completed, add enough sterile water to the white porcelain plate, then take the gel out of the gun box, place it on the white porcelain plate, and take photos. After recording, the gel can be stored in sterile water or water containing 20% ​​glycerol for a short period of time, or it can be made into dry gel for long-term storage.

[0138] Results: The protein molecular weight of oocyte extracts obtained from different batches of crucian carp was detected by Coomassie Brilliant Blue staining. Fig. 9 .Depend on Fig. 9 It can be seen that the relative molecular weight of the protein in the crucian carp oocyte extract is basically between 8kDa-130kDa. Among them, most of the proteins are concentrated between 20kDa-30kDa and 58kDa-130kDa. There are fewer or no protein bands in the remaining intervals, indicating that the extraction process used in the oocyte extract of the present invention is stable.

Claims

1. Application of Smad6 gene in promoting reprogramming of fibroblasts into stem cells, characterized in that: By overexpressing the Smad6 gene to inhibit Smad2 / 3 activation, the upregulation of important transcription factors C-Myc, Nanog and Oct4 that maintain and regulate the stem cell state is promoted, thereby promoting the reprogramming of fibroblasts into stem cells, specifically promoting the reprogramming of fibroblasts transfected with fish oocyte extract into stem cells.

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