CN113549593B - Pharmaceutical preparation for promoting proliferation of epidermal stem cells - Google Patents
Pharmaceutical preparation for promoting proliferation of epidermal stem cells Download PDFInfo
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Abstract
The invention provides a pharmaceutical preparation for promoting proliferation of epidermal stem cells, and belongs to the technical field of epidermal stem cells. The pharmaceutical preparation contains effective dose of lnc-XPNPEP1-1:1 siRNA or nucleic acid sequence modifier and pharmaceutically acceptable carrier. The use of the pharmaceutical preparation can obviously accelerate the proliferation rate of cells, increase the expression quantity of the Cyclin-D1, and reduce the expression quantity of the Cyclin-inhibiting gene P21.
Description
Technical Field
The invention belongs to the technical field of epidermal stem cells, and particularly relates to a pharmaceutical preparation for promoting proliferation of epidermal stem cells.
Background
The epidermal stem cells (epidermal stem cells) are cell groups with unlimited proliferation capability and proliferation and differentiation into various functions in epidermis, and are ideal seed cells for preparing tissue engineering skin. The epidermal cells play an important role in the processes of epidermal regeneration, skin homeostasis maintenance and wound repair.
Various wounds and injuries can cause damage to the superficial skin, not only resulting in localized exudation, infection and pain, but severe patients can experience systemic infection, electrolyte imbalance, shock, and even death in some patients. As seed cells of the skin, the epidermal stem cells open up a new therapeutic approach for skin repair and reconstruction. When damage occurs, epidermal stem cells can effect repair of the skin by proliferation and migration. Therefore, by means of gene therapy, transfection of the epidermal stem cells to increase proliferation and migration of the epidermal stem cells can make the epidermal stem cells better play a role in repairing skin injury.
RNA interference refers to the phenomenon of highly conserved, highly efficient, specific degradation of homologous mRNA induced by double stranded RNA during evolution. Since the expression of a specific gene can be specifically knocked out or shut down using RNAi technology, this technology has been studied by functional genomics, and a part of RNA drugs have entered the clinical trial stage.
Disclosure of Invention
The invention aims to provide a pharmaceutical preparation for promoting proliferation of epidermal stem cells.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a pharmaceutical preparation for promoting the proliferation of epidermal stem cells, which contains an effective dose of lnc-XPNPEP1-1:1 siRNA or a nucleic acid sequence modifier thereof and a pharmaceutically acceptable carrier.
Preferably, the sequence of the transcript of the lnc-XPNPEP1-1:1 is shown in SEQ ID NO. 1.
Preferably, the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the nucleic acid sequence modification is obtained by ribose modification or base modification of any nucleotide based on SEQ ID NO.4 or SEQ ID NO. 5.
Preferably, the carrier is a liposome, nanoparticle, cholesterol or virus.
Furthermore, the invention provides application of an inhibitor of lnc-XPNPEP1-1:1 in preparing a pharmaceutical preparation for promoting proliferation of epidermal stem cells, wherein the transcript sequence of lnc-XPNPEP1-1:1 is shown as SEQ ID NO. 1.
Preferably, the inhibitor is an effective dose of an siRNA of lnc-XPNPEP1-1:1 or a nucleic acid modification thereof.
Preferably, the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5; the nucleic acid sequence modifier is obtained by ribose modification or base modification of any nucleotide on the basis of SEQ ID NO.4 or SEQ ID NO. 5.
Furthermore, the invention provides application of an inhibitor of lnc-XPNPEP1-1:1 in preparing a Cyclin-D1 gene expression promoter or a Cyclin P21 gene expression inhibitor, wherein the transcript sequence of lnc-XPNPEP1-1:1 is shown as SEQ ID NO. 1.
Preferably, the inhibitor is an effective dose of lnc-XPNPEP1-1:1 siRNA or nucleic acid modifications thereof; the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5; the nucleic acid sequence modifier is obtained by ribose modification or base modification of any nucleotide on the basis of SEQ ID NO.4 or SEQ ID NO. 5.
The beneficial effects of the invention are as follows:
the invention provides an siRNA of lnc-XPNPEP1-1:1 capable of effectively promoting proliferation of epidermal stem cells, which is used for preparing a pharmaceutical preparation for promoting proliferation of epidermal stem cells and realizing rapid large-scale expansion of the epidermal stem cells, wherein after the epidermal stem cells are transfected with the siRNA, the proliferation rate of the cells is obviously accelerated, the expression quantity of a Cyclin-D1 gene is increased, and the expression quantity of a Cyclin-inhibiting gene P21 is reduced.
Drawings
FIG. 1 shows the inhibitory effect of synthesized siRNA on lnc-XPNPEP1-1:1, where P <0.001 is expressed, indicating that the difference is statistically significant;
after the MTT test of FIG. 2 shows that lnc-XPNPEP1-1:1 is inhibited, proliferation of epidermal stem cells is changed, wherein P is less than 0.05, P is less than 0.01, and P is less than 0.001, so that the difference has statistical significance;
FIG. 3 shows changes in mRNA expression of Cyclin P21 and Cyclin D1 after inhibition of lnc-XPNPEP1-1:1 by quantitative PCR, where P <0.05 and P <0.01 indicate that the differences are statistically significant;
FIG. 4 cell scratch assay to detect migration of cells after lnc-XPNPEP1-1:1 is inhibited;
FIG. 5 Western Blot detection of changes in stromal cell derived factor-1 (SDF-1) and chemokine receptor 4 (CXCR 4) after lnc-XPNPEP1-1:1 is inhibited.
Detailed Description
In order to clearly illustrate the technical characteristics of the scheme, the scheme is explained below through a specific embodiment. It should be noted that the examples of the present invention are only for illustrating the present invention and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of human skin epidermal Stem cells
(1) Removing subcutaneous adipose tissue from skin tissue and culture dish, and washing with PBS containing double antibodies for 3 times for 5min each time;
(2) Cutting skin tissue into skin fragments, adding 3ml of 0.25% protease, placing the skin with epidermis facing upwards and dermis facing downwards in a culture dish, and standing in a refrigerator at 4deg.C for digestion overnight;
(3) Taking out after overnight, separating epidermis and dermis with forceps, cutting epidermis with a scalpel, placing in a 6cm petri dish, adding 2ml trypsin, and placing in a cell incubator for digestion for 15min;
(4) Digestion was terminated by adding 2ml DMEM medium containing 10% FBS;
(5) Repeatedly blowing by using a liquid-transferring gun to fully release the epidermal cells;
(6) Filtering the cell suspension by using a 200-mesh filter screen, and centrifuging the filtered cell filtrate by using a centrifugal machine 290g for 5min;
(7) Removing the supernatant, adding an epidermal stem cell culture medium KGM2 to resuspend the cells and counting the cells;
(8) Per cm 2 Cells were seeded into collagen IV coated cell culture dishes at a density of 2 ten thousand cells;
(9) Changing the culture medium for 10min, removing cells which are not adhered to the culture medium, culturing for 2-3 days, and changing the liquid;
(10) When the cell growth reaches about 80%, 0.25% trypsin is used for digestion, and the human skin epidermal stem cells are obtained and used for subsequent experiments.
EXAMPLE 2 siRNA Synthesis and cell transfection
1. Design of synthetic siRNA molecules
According to the sequence of the lnc-XPNPEP1-1:1 (SEQ ID NO. 1), 3 siRNA sequences targeting the lnc-XPNPEP1-1:1 gene are designed and synthesized, and the specific sequences are as follows:
siRNA-1
sense strand: 5 '. Fwdarw.3': AUUAACUUCCUUAGGAUUC (SEQ ID NO. 2);
antisense strand: 5 '. Fwdarw.3': GAAUCCUAAGGAAGUUAAU (SEQ ID NO. 3);
siRNA-2
sense strand: 5 '. Fwdarw.3': UAAUUUGGCAUUACAAUAG (SEQ ID NO. 4)
Antisense strand: 5 '. Fwdarw.3': CUAUUGUAAUGCCAAAUUA (SEQ ID NO. 5)
siRNA-3
Sense strand: 5 '. Fwdarw.3': UGAAUCUGUUUUAAUUCAC (SEQ ID NO. 6)
Antisense strand: 5 '. Fwdarw.3': GUGAAUUAAAACAGAUUCA (SEQ ID NO. 7)
Secondly, siNC which is different from the human gene is designed and synthesized as a negative control, and the specific sequence is as follows:
siNC
sense strand: 5 '. Fwdarw.3': UAACACAUGGCUGAAUUUC (SEQ ID NO. 8)
Antisense strand: 5 '. Fwdarw.3': GAAAUUCAGCCAUGUGUUA (SEQ ID NO. 9)
2. Transfection of siRNA
(1) The epidermal stem cells obtained in the example 1 are paved in a 6-pore plate and placed in a cell incubator for overnight culture;
(2) After overnight, the medium was removed, siRNA-1, siRNA-2, siRNA-3 and siNC were transfected into epidermal stem cells according to Lipofecatamine2000 instructions, and cells that were not transfected in normal culture were set as a control group.
Example 3 RNA extraction experiments
(1) After 48 hours of cell transfection, the culture medium is removed, 1ml of Trizol is added, and after repeated blowing and cell lysis, the cells are kept stand for 5 minutes at room temperature;
(2) Adding 200ul chloroform, gently mixing for 15s, standing at room temperature for 10min,12000rpm, 4 ℃, centrifuging for 15min;
(3) Carefully transfer the supernatant to a new EP tube, add an equal volume of isopropanol, rest in ice bath for 10min,12000rpm, 4℃and centrifuge for 5min;
(4) Removing the supernatant, adding 1ml of 75% ethanol, washing for 2 times, gently shaking, and centrifuging at 1200rpm and 4 ℃ for 5min;
(5) Removing the supernatant, airing, adding DEPC water, and standing at-80 ℃ for standby.
EXAMPLE 4 reverse transcription Using reverse transcription kit
1. Removal of genomic DNA
(1) The following reaction solutions were prepared on ice:
| component (A) | Volume of |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1.0μg |
| RNase Free dH2O | Up to 10μl |
(2) The reaction solution was placed in a PCR apparatus at 42℃for 2min, and then placed on ice.
2. Reverse transcription
(1) Preparing the reaction solution on ice
| Component (A) | Volume (mul) |
| Reaction solution for removing genome DNA | 10 |
| PrimeScript |
1.0 |
| RT Primer Mix | 1.0 |
| 5×PrimeScript Buffer 2 (for Real Time) | 4.0 |
| RNase Free dH2O | 4.0 |
| Total | 20 |
(2) The reaction solution was placed in a PCR apparatus and reacted at 37℃for 15min and 85℃for 5 seconds.
Example 5 fluorescent quantitative PCR reaction
(1) The following reaction system was configured on ice:
| component (A) | Volume (mul) |
| Hieff ® qPCR SYBR Green Master Mix (Low Rox Plus) | 10 |
| Forward Primer (10μM) | 0.4 |
| Reverse Primer (10μM) | 0.4 |
| |
1 |
| Sterile ultrapure water | To 20 |
(2) The PCR reactions were performed according to the following table procedure:
| circulation step | Temperature (temperature) | Time | Cycle number | |
| Pre-denaturation | 95 | 5min | 1 | |
| Denaturation (denaturation) | 95℃ | 10s | 40 | |
| Annealing/extension | 62℃ | 30s |
(3) Primer sequences
(2) Results Using 2 -△△Ct The method was used for analysis.
As shown in FIG. 1, the relative expression level of lnc-XPNPEP1-1:1 of siRNA-1 is 0.298+ -0.029, and the difference from control is statistically significant;
the relative expression level of lnc-XPNPEP1-1:1 of siRNA-2 is 0.197+/-0.046, and the difference from control has statistical significance;
the relative expression level of lnc-XPNPEP1-1:1 of siRNA-3 is 0.312+/-0.053, and the difference from control has statistical significance;
the relative expression level of lnc-XPNPEP1-1:1 of si-NC is 0.989+ -0.052, and the difference from control is not statistically significant;
from the experimental results, the 3 siRNAs provided by the invention can effectively inhibit the expression of lnc-XPNPEP1-1:1, wherein the inhibition effect of siRNA-2 is most remarkable.
Example 6
MTT test detects proliferation change condition of epidermal stem cells after lnc-XPNPEP1-1:1 is inhibited
(1) Taking 100ul 2X 10 3 The epidermal stem cell cells growing logarithmically are inoculated in a 96-well plate pre-coated with type IV collagen;
(2) After 2d incubation, the medium was removed and siNC and siRNA-2 were transfected into epidermal stem cells according to lipofectamine 2000 instructions;
(3) At 1-4d after transfection, the absorbance at 490nm was measured by incubation in MTT cell incubator for 4h, respectively.
The results of the experiment are shown in fig. 2:
at 1d, the OD value of the siNC is 0.185+/-0.013, the OD value of the siRNA-1 is 0.221+/-0.015, and the P=0.035, and the difference has statistical significance;
at 2d, the OD of siNC was 0.233+ -0.014, the OD of siRNA-1 was 0.381+ -0.024, P=0.0008, the difference was statistically significant;
at 3d, the OD value of siNC is 0.307+ -0.012, the OD value of siRNA-1 is 0.490+ -0.045, and P=0.0023, the difference is statistically significant;
at 4d, the OD of siNC was 0.390+ -0.025, the OD of siRNA-1 was 0.547+ -0.025, and P=0.0015, the differences were statistically significant;
from the experimental results, the proliferation of the epidermal stem cells can be effectively promoted after the lnc-XPNPEP1-1:1 is inhibited.
Example 7
Quantitative PCR detection of changes in mRNA expression of Cyclin P21 and Cyclin D1 after lnc-XPNPEP1-1:1 was inhibited
(1) The epidermal stem cells obtained in the example 1 are paved in a 6-pore plate and placed in a cell incubator for overnight culture;
(2) After overnight, the medium was removed and siNC and siRNA-2 were transfected into epidermal stem cells according to lipofectamine 2000 instructions;
(3) After 48h of transfection, removing the culture medium, extracting RNA, and detecting the expression conditions of cell cycle related genes Cyclin-D1 and P21;
(4) Primer sequences of Cyclin-D1 and P21 are shown in the following table, and PCR reaction conditions are the same as in example 5;
the experimental result shows that the relative expression quantity of Cyclin-D1 of siRNA-1 is 1.447 + -0.078, P=0.0013, and the difference has statistical significance;
the relative expression amount of P21 of siRNA-1 is 0.715+/-0.072, P=0.0122, and the difference has statistical significance;
from the above experimental results, it can be seen that after lnc-XPNPEP1-1:1 is inhibited, the expression level of Cyclin-D1 promoting the cell cycle is significantly increased, and the expression level of P21 inhibiting the cell cycle is significantly decreased.
Example 8 cell scratch assay detection of migration of cells after lnc-XPNPEP1-1:1 was inhibited
(1) The epidermal stem cells obtained in the example 1 are paved in a 6-pore plate and placed in a cell incubator for overnight culture;
(2) After overnight, the medium was removed and siNC and siRNA-2 were transfected into epidermal stem cells according to lipofectamine 2000 instructions;
(3) When the confluence of cells reaches about 90%, a straight line is drawn at the center of the hole by using the gun head of a 200ul pipette;
(4) The floating cells were removed using PBS, after 24h, observed and photographed under an inverted microscope.
As shown in the experimental results in FIG. 4, it can be seen that the migration ability of the epidermal stem cells is significantly enhanced compared with the control group after the lnc-XPNPEP1-1:1 is inhibited.
Example 9
Western Blot detection of changes in stromal cell derived factor-1 (SDF-1) and chemokine receptor 4 (CXCR 4) D after lnc-XPNPEP1-1:1 is inhibited
(1) The epidermal stem cells obtained in the example 1 are paved in a 6-pore plate and placed in a cell incubator for overnight culture;
(2) After overnight, the medium was removed and siNC and siRNA-2 were transfected into epidermal stem cells according to lipofectamine 2000 instructions;
(3) After 48h of transfection, the medium was removed, protein lysate was added, cells were scraped off using a cell scraper, and transferred to a 1.5ml EP tube;
(4) Placing the EP tube in an ice box, placing the ice box on a shaking table, and fully cracking for 30min;
(5) After centrifugation at 12000rpm,4℃for 10min, the supernatant was transferred to a new EP tube;
(6) Absorbing 2ul of lysate, quantifying protein by BCA method, adding loading buffer to adjust protein sample concentration to 2 μg/μl, and decocting in boiling water for 5min;
(7) Preparing separating gel and concentrating gel, loading, assembling electrophoresis tank, and electrophoresis at 80V for 20min until bromophenol blue reaches bottom of gel.
(8) Assembling an electric rotating clamp according to a classical sandwich model, installing an electric rotating groove, and rotating a film for 90min at 250 mA;
(9) After the electric conversion is finished, the membrane is taken out and placed in 5% of skimmed milk powder, placed on a shaking table and sealed for 1h at room temperature;
(10) After washing the membrane with TBST, adding a primary antibody, and incubating at 4 ℃ overnight;
(11) Recovering the primary antibody, washing the membrane by using TBST, adding the secondary antibody, placing the membrane on a shaking table, and incubating for 1.5h at room temperature;
(12) Development exposure and photographing are performed.
As shown in FIG. 5, it can be seen that the protein expression levels of SDF-1 and CXCR4 in the epidermal stem cells are significantly increased after the lnc-XPNPEP1-1:1 is inhibited. It was demonstrated that inhibition of lnc-XPNPEP1-1:1 is effective in promoting protein expression of SDF-1 and CXCR4, thereby promoting cell migration.
Sequence listing
<110> Shandong gold biological development Co., ltd
<120> a pharmaceutical preparation for promoting proliferation of epidermal stem cells
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Claims (4)
1. A pharmaceutical formulation for promoting epidermal stem cell proliferation, comprising an effective dose of lnc-XPNPEP1-1:1 siRNA and a pharmaceutically acceptable carrier;
the transcript sequence of the lnc-XPNPEP1-1:1 is shown in SEQ ID NO. 1;
the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5.
2. The pharmaceutical formulation of claim 1, wherein the carrier is a liposome, nanoparticle, cholesterol, or virus.
Use of an inhibitor of lnc-XPNPEP1-1:1 for the preparation of a pharmaceutical formulation for promoting proliferation of epidermal stem cells, characterized in that the transcript sequence of lnc-XPNPEP1-1:1 is shown in SEQ ID No. 1;
the inhibitor is an effective dose of siRNA of lnc-XPNPEP 1-1:1;
the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5.
The use of an inhibitor of lnc-XPNPEP1-1:1 in the preparation of a Cyclin-D1 gene expression promoter or an inhibitor of Cyclin P21 gene expression, wherein the transcript sequence of lnc-XPNPEP1-1:1 is shown in SEQ ID No. 1;
the inhibitor is an effective dose of siRNA of lnc-XPNPEP 1-1:1; the sequences of the siRNA are shown as SEQ ID NO.4 and SEQ ID NO. 5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005245274A (en) * | 2004-03-03 | 2005-09-15 | Zoegene Corp | Cell proliferation promoter |
| CN104561101A (en) * | 2014-12-22 | 2015-04-29 | 中国人民解放军第二军医大学 | Method and application of MicroRNA (micro ribonucleic acid) 221-3p in preparation of epidermal cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005245274A (en) * | 2004-03-03 | 2005-09-15 | Zoegene Corp | Cell proliferation promoter |
| CN104561101A (en) * | 2014-12-22 | 2015-04-29 | 中国人民解放军第二军医大学 | Method and application of MicroRNA (micro ribonucleic acid) 221-3p in preparation of epidermal cells |
Non-Patent Citations (2)
| Title |
|---|
| The RNA-binding protein YBX1 regulates epidermal progenitors at a posttranscriptional level;Eunjeong Kwon et al;Nature Communications;第1-16页 * |
| 基质细胞衍生因子-1对表皮干细胞的促增殖作用;王瑶等;标记免疫分析与临床;全文 * |
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Address after: Room 401, Shandong Stem Cell and Regenerative Medicine Technology Innovation Center, Daxue Road, Changqing District, Jinan City, Shandong Province, 250000 (the original physical examination building in the university town of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine) Patentee after: Shandong Kejin Biological Development Co.,Ltd. Address before: 250000 room 1602, block D, Golden Times Square, No. 9999, Jingshi Road, Jinan area, China (Shandong) free trade Experimental Zone, Jinan City, Shandong Province Patentee before: Shandong Kejin Biological Development Co.,Ltd. |