Inhibitor for inducing pluripotent stem cells, and inducing method and application thereof

[0059] Example 1 MicroRNA-421 inhibitor induces iPS cells

[0060] In this example, cardiac fibroblasts were isolated from mouse heart tissue, and a microRNA-421 inhibitor was used to induce the cardiac fibroblasts to produce iPS cells. The specific experimental operation steps are as follows:

[0061] Take adult mice, open the chest under aseptic conditions, cut 3 hearts, rinse the hearts with pH 7.4 PBS three times, then place them on ice, add 1 mL of DMEM with 0.08% pancreatin and 1% collagenase for digestion 50mL of DMEM digestion solution contains 2mL of 0.08% pancreatin and 2mL of 1% collagenase II (trypsin and collagenase II were ordered from Beijing Xinjingke Biotechnology Company and sigma Company) First use scissors and blades to cut the tissue Chop into 1-2mm pieces. Then add 5mL digestion solution (50mL DMEM digestion solution contains 2mL of 0.08% trypsin and 2mL of 1% collagenase II) for digestion, and digest in a shaker at a constant temperature for 4-6 minutes at 37°C (the shaker speed is 60r/ min), then use a pipette tip to repeatedly blow twice at a constant speed, let stand for 2 minutes at room temperature, transfer the supernatant to a new 50 mL centrifuge tube (pre-add 2 mL of FBS), and then digest the remaining tissue blocks in the same way , Collect and combine all the supernatants, centrifuge at 800 rpm/min for 10 minutes, discard the supernatant, wash once with 20% FBS in DMEM medium, after centrifugation, remove the supernatant, and incubate with an appropriate amount of 8-10 mL of DMEM containing 20% ​​FBS Resuspend the cells in the solution, screened by a 200-mesh cell sieve, mix them and spread them on a 10cm petri dish, and place them in an incubator containing 37℃ in 5% CO 2 Under cultivation. After 2 hours, change the medium: At this time, the fibroblasts adhere to the wall faster and the cardiomyocytes adhere slowly. After the medium is changed, the culture dish is mainly composed of myocardial fibroblasts, and then culture overnight until the cell length is 3-4 days. After it is full, it is used for induction experiments.

[0062] Prepare a 6-well plate, spread 0.1% gelatin (weigh 0.1g of gelatin, dissolve it in 100mL of water, filter it with a 0.25μm cell filter) on a 6-well cell culture plate, and place it in a clean bench to dry , And then passage the myocardial fibroblast cells to a 6-well plate, using 20% ​​DMEM medium, placed in 5% CO 2 Incubate in a 37℃ incubator for 36 hours, add 160nM microRNA-421 inhibitor (sequence 5'-GCGCCCAAUUAAUGUCUGUUGAU-3' to each well, and the base ribose is 2'-methoxylated modified, commissioned by Shanghai Jima Pharmaceutical Technology Co., Ltd. synthesis). To induce the generation of iPS cells, see Kazutoshi Takahashi et al., 2007, Nature protocols, 2(12):3081-3089 for specific experimental procedures. Induce the production of multifunctional stem cells, namely mi-iPS cells, this group is the experimental group (see the experimental results figure 1 B), the control group is a negative control with microRNA-421 inhibitor added (see figure 1 A).

[0063] figure 2 A is the comparison of the number of iPSC clones induced by microRNA-421 inhibitor and the number of iPSC clones induced by four viral factors; 1 in the figure is the number of iPSC clones induced by four factors: the four factors induce iPSC by adding OCT4 , KLF4, SOX2, C-MYC four factors of virus (virus is packaged by the vector, the steps are as follows. The vector was purchased from Addgene Biological Company) induced. The virus of the four factors was co-transfected with the Fugene HD transfect reagent (purchased from Roche Biological Company) when the 293T cells grew to 90%. The virus supernatant was collected after 48 hours. Four factor viruses were obtained (Kazutoshi Takahashi et al. 2007). Figure 2 shows the iPSC clone formation induced by microRNA-421 inhibitor.

[0064] The induction test is the same as the experimental conditions in Figure 1A. Through the statistics of the number of iPSC clones formed in the two sets of experiments, it is found that the number of iPSC clones in the experimental group with microRNA-421 inhibitors is significantly increased (see figure 2 A).

[0065] figure 2 B is the luciferase detection experiment to detect the regulation of microRNA-421 on the 3'-UTR region of the four factors (for the experimental procedure, refer to the Promega Dual-luciferase reporter assay system kit, the item number is TM040). figure 2 B. The abscissa 1 is the control group, and the microRNA-421 expression vector and pRL-tk internal reference plasmid are added, and the ratio of firefly luciferase to Renilla luciferase activity is very low; 2 is the negative control group, with microRNA-421 added The ratio of firefly luciferase to Renilla luciferase activity measured after expression vector, pRL-tk internal reference plasmid and pGL3 empty vector (without adding four factors); figure 2 3 in B represents the experimental group with the pRL-tk internal reference plasmid, microRNA-421 expression vector and pGL3-OCT4 vector; 4 in the figure represents the experiment with the pRL-tk internal reference plasmid, microRNA-421 expression vector and pGL3-KLF4 vector Group; Figure 5 represents the experimental group added pRL-tk internal reference plasmid, microRNA-421 expression vector and pGL3-SOX2 vector; Figure 6 represents the addition of pRL-tk internal reference plasmid, microRNA-421 expression vector and pGL3-C- Experimental group of MYC vector. figure 2 The ratio of 3-6 firefly luciferase to Renilla luciferase activity in B is significantly reduced. miRNA-421 inhibits the expression of the four genes by inhibiting the 3'-UTR region of OCT4, KLF4, SOX2, C-MYC genes . Experiments confirmed that miRNA-421 simultaneously regulates the four totipotent factors of OCT4, KLF4, SOX2, and C-MYC.

[0066] Example 2 Preparation and identification of mouse myocardial infarction model

[0067] In this example, a mouse myocardial infarction model was prepared and identified.

[0068] 1) The left coronary artery occlusion method was used to induce myocardial infarction in mice, thereby establishing a mouse myocardial infarction model.

[0069] After anesthesia, the mice were fixed on the experimental wooden board. ECG monitoring. Cut the trachea in the middle of the neck and intubate, connect the animal artificial ventilator, open the chest in the fourth intercostal space, carefully lift the pericardium and cut open, fully expose the heart and blood vessels. Between the pulmonary artery cone and the left atrial appendage, using the trunk of the left coronary vein as a mark, insert the needle below the root of the left atrial appendage with a depth of 0.5mm; pass the 6/0 suture through the surface of the myocardium and exit next to the pulmonary artery Needle; After the ECG is stable, the left anterior descending coronary artery (LAD) is ligated. The ST segment arch dorsal elevation in leads I and aVL is greater than 0.1mV and lasts for more than 0.5h as a sign of successful ligation. Clear the blood in the chest cavity and close the chest cavity with aspirating gas with a needle-removing syringe.

[0070] After a certain period of time (in this experiment, postoperative ischemia 45min reperfusion was used) reperfusion was used to prepare a mouse myocardial infarction model (mouse ischemia-reperfusion model referred to as I/R model).

[0071] 2) Methods to identify and detect cardiac function in mouse models of myocardial infarction:

[0072] (1) Determine the area of ​​myocardial infarction:

[0073] Cut the heart specimen, cut the left ventricle into thin slices (7μm) evenly along the longitudinal axis of the heart, and place it in NBT solution (nitrotetrazolium blue chloride: 0.06133gNBT with phosphate buffer solution to make the volume to 100ml, store in the dark) 37 ℃ water bath for 15 minutes. The necrotic area is not stained because there is no dehydrogenase, and the non-necrotic area is stained blue because it contains dehydrogenase. Under a dissecting microscope, carefully separate the stained area from the unstained area and weigh them with an electronic balance. The size of the infarct area is expressed as the percentage of the weight of the infarct area and the weight of the left ventricle.

[0074] (2) Determine the apoptosis of cardiomyocytes:

[0075] TUNEL and Cell Death Detection ELISA were used to detect cardiomyocyte apoptosis.

[0076] TUNEL method to detect cardiomyocyte apoptosis: digestion with proteinase K (digested samples are tissue sections taken from the heart of myocardial infarction model mice to observe cardiomyocyte apoptosis. The concentration of proteinase K is 20μg/mL, and 10mM Tris- Dissolved in HCl, pH 7.4-8.0, purchased from Hoffmann-La Roche Co., Ltd. After deparaffinization of the tissue section, digestion with proteinase K, and the sample is placed at 21℃-37℃ for 15min-30min) to expose the DNA. Terminal transferase (terminal transferase: Terminal transferase, TdT (purchased from Biyuntian Institute of Biotechnology), is a DNA polymerase that does not require a template, catalyzes the binding of deoxynucleotides to the 3'hydroxyl end of DNA molecules. Protruding, recessed or smooth-ended single- and double-stranded DNA molecules can be used as the substrate of TdT) under the mediation of dUTP, in which fluorescein-labeled dUTP is incorporated into the 3'OH of the double-stranded or single-stranded DNA of apoptotic cells End (use the TUNEL cell apoptosis detection kit, follow the product instructions provided by the kit manufacturer, the kit is used to detect the nuclear DNA breakage of the tissue cells in the early process of apoptosis. The principle is fluorescein labeling Under the action of deoxyribonucleotide terminal transferase, the dUTP can be connected to the 3'-OH end of broken DNA in apoptotic cells and specifically bind to the luciferin antibody linked to horseradish peroxidase, which in turn It reacts with HRP substrate diaminobenzidine to produce a strong color reaction, which is dark brown, and specifically and accurately locates apoptotic cells, so apoptotic cells can be observed under an optical microscope; due to normal or proliferating cells There is almost no DNA fragmentation, so there is no 3'-OH formation, and it is rarely stained.), observe the cells labeled with fluorescein-dUTP under a fluorescence microscope (the laser confocal microscope was used in the experiment, and the excitation wavelength of the photo was taken. It is 790nm, magnified 100 times). The apoptotic cell nucleus is green fluorescence, and the normal cell nucleus is blue fluorescence.

[0077] Calculate the apoptosis index (AI) of each group, AI=number of apoptotic cells/total number of cells (each high-powered field of view), take 5 high-powered field of view for each slide, and take the average value. Prepare tissue sections, and then perform TUNEL staining. Use a laser confocal microscope to observe and photograph the apoptosis status, perform statistics, and calculate the apoptosis index according to AI=number of apoptotic cells/total number of cells.

[0078] (3) Determination of heart function:

[0079] There are two methods of detecting heart function:

[0080] 1. Determination of echocardiographic parameters: M-mode echocardiography examines the fundus and ventricular complexes to determine the left atrial diameter (LA), left ventricular end diastolic diameter (LVIDd), left ventricular end systolic diameter (LVIDs), ventricular Interval thickness (IVSd). Calculate stroke volume (SV), volume per minute (CO), left ventricular ejection fraction (EF) and short axis shortening rate (FS%), left ventricular short axis shortening rate (FS%) is left ventricular contraction Function index parameters). The short axis shortening rate of the left ventricle, FS%, is directly proportional to the left ventricular systolic function of the heart, which can directly indicate the strength of the heart.

[0081] FS%=[(LVIDd-LVIDs)/LVIDd]×100

[0082] 2. On the apical four-chamber view, place the sampling volume at the mitral valve orifice and the left ventricular outflow tract respectively, measure the early diastolic blood flow velocity E peak of the mitral valve orifice, and the late diastolic blood flow velocity A peak and calculate the E peak /A peak ratio. The tested sample was injected with 1×10 6 A myocardial infarction model mouse with mi-iPS cells/64mg/kg microRNA-421 inhibitor was measured with echocardiography for peak E and peak A. Combined with electrocardiogram to measure left ventricular isovolumic relaxation time (IVRT), left ventricular isovolumic contraction time (IVCT), left ventricular ejection time (ET). Calculate Tei index=IVRT+IVCT/ET. The measurement data is measured for 3 consecutive cardiac cycles, and the average value is taken. Tei index, also known as Myocardial Performance Index (MPI), can comprehensively reflect the diastolic function of the ventricular systolic machine, and is mainly used to study the left ventricular function of dilated cardiomyopathy.

[0083] In this paper, the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter (LVIDs) were used to calculate the left ventricular short axis shortening rate FS% to detect the degree of repair of myocardial infarction and cardiac function in mice before and after administration.

[0084] Example 3 Using mi-iPS cells to treat myocardial infarction in mice, observe the changes in the area of ​​myocardial infarction and the degree of cardiac function repair in mice.

[0085] Six-week-old C57 mice were selected, and the myocardial infarction model mice were prepared according to the step (1) of Example 2. Five mice were used as the control group and five mice were used as the experimental group. When the model mice were prepared for 24 hours, the mice in the experimental group were injected with 1×10 at the myocardial infarction. 6 Mi-iPS cells. One week later and one month later, the area of ​​myocardial infarction in mice was changed and the degree of repair of myocardial function in mice was measured with a supersonic cardiograph. The change of mouse heart function (FS% value) after one month see image 3.

[0086] image 3 The data provided in is an observation after one month.