CN106119375B - Medicine with regulating and controlling effect on articular cartilage degeneration and verification method - Google Patents
Medicine with regulating and controlling effect on articular cartilage degeneration and verification method Download PDFInfo
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- CN106119375B CN106119375B CN201610525458.8A CN201610525458A CN106119375B CN 106119375 B CN106119375 B CN 106119375B CN 201610525458 A CN201610525458 A CN 201610525458A CN 106119375 B CN106119375 B CN 106119375B
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Abstract
The invention discloses a medicament for regulating and controlling articular cartilage degeneration and a verification method, wherein the medicament for regulating and controlling articular cartilage degeneration is miRNA-140, and the regulation and control of the articular cavity injection miRNA-140 on rat knee joint cartilage degeneration, the regulation and control of the medicament in osteoarthritis cartilage cells and the regulation and control of the medicament in osteoarthritis joint fluid are verified. The invention separates and cultures human normal and different degree OA chondrocytes in vitro, the expression quantity of the OA chondrocytes is gradually reduced along with the increasing of the OA degree, the expression in the severe OA chondrocytes is the lowest, the expression of miRNA-140in OA joint fluid is obviously reduced compared with that of a normal group, the loss of Collagen II protein in joint cartilage tissues can be obviously slowed down by injecting miRNA-140agomir in a joint cavity, meanwhile, the expression of MMP13 and ADAMTS-5 protein is obviously inhibited, and the degradation of rat joint cartilage is obviously delayed.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a medicine with a regulating and controlling effect on articular cartilage degeneration and a verification method.
Background
Osteoarthritis (OA) is a common disease characterized mainly by progressive articular cartilage degeneration, and the clinical manifestations mainly include progressively aggravated joint pain, deformity, and movement disorder. The incidence rate, teratogenicity and disability rate of OA are high, but the pathogenesis of OA is still unclear and an effective treatment means is lacking. In recent years, research shows that microRNA (miRNA) can be combined with target gene (mRNA) to cause the degradation or translational inhibition of the latter, regulate the expression of mRNA about 1/3 in mammals, and play an important role in cell proliferation, differentiation, senescence, apoptosis and the like. miRNA-140 has cartilage specificity, is stably expressed in human normal articular cartilage, and plays an important role in regulating and controlling normal homeostasis and functions of articular cartilage. There is still controversy about the expression rule of miRNA-140in the process of OA, but it is generally considered that miRNA-140 plays an important role in protecting articular cartilage during the development of OA. Therefore, miRNA-140 can be used as an entry point for treating OA, but at present, related researches are mainly in vitro chondrocyte experiments or gene knockout/transgenic animal experiments, and the specific method and the way guidance for deeply researching the miRNA-140 to treat OA are limited.
Disclosure of Invention
The invention aims to provide a medicine with a regulation and control effect on articular cartilage degeneration and a verification method, and aims to solve the problems that the specific method and the path guidance effect of the existing miRNA-140 for treating OA are limited.
The invention is realized in such a way that the medicine with the function of regulating the degeneration of the articular cartilage is miRNA-140.
The invention also aims to provide a method for verifying the regulation and control effect of miRNA-140in osteoarthritis chondrocytes, which comprises the following steps:
Firstly, verifying the expression rule of miRNA-140in human normal and Osteoarthritis (OA) chondrocytes;
Secondly, screening the optimal condition of the miRNA-140 for transfecting the human chondrocytes;
Finally, the regulation and control function and mechanism of the miRNA-140 expression up-regulation or down-regulation on human OA chondrocytes are verified.
Further, the method for verifying the expression rule and the regulation and control action of miRNA-140in osteoarthritis chondrocytes specifically comprises the following steps:
(1) According to the Classification standard of Kellgren and Lawrence (KL) OA imaging science, collecting human normal (KL 0 grade), light (KL1-2 grade), medium (KL 3 grade) and heavy (KL 4 grade) OA fresh cartilage specimens, and separating, culturing and identifying chondrocytes in vitro;
(2) Detecting the expression level of miRNA-140in normal and OA chondrocytes by adopting a fluorescent quantitative PCR method, and analyzing the expression change rule of the miRNA-140;
(3) transfecting chondrocytes by miRNA-140mimic with different concentrations (0, 25, 50, 100 and 200nM), detecting the expression levels of cells Collagen II and MMP13 genes and proteins by PCR and Western blotting methods at different time points (24, 48 and 72h) after transfection, and screening the optimal concentration and the optimal time for detecting the effect of the miRNA-140mimic transfected chondrocytes;
(4) The miRNA-140 micic or miRNA-140inhibitor with the optimal concentration is adopted to transfect normal and OA chondrocytes, the expression levels of genes and proteins of cells Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 are detected at the optimal time, the expression differences of the genes and the proteins in various groups of chondrocytes after the miRNA-140 is up-regulated or down-regulated are compared, and the regulation and control effect and the molecular mechanism of the miRNA-140 on the OA chondrocytes are analyzed.
The invention also aims to provide a method for verifying the regulation and control effect of miRNA-140in osteoarthritis joint fluid, which comprises the following steps:
Detecting the expression rule of miRNA-140in human normal and OA joint fluid, and verifying the correlation between the expression level of miRNA-140 and the severity of OA.
Further, the method for verifying the regulation and control effect of the miRNA-140in osteoarthritis joint fluid specifically comprises the following steps:
Collecting 10 cases of knee joint fluid specimens of normal, light, medium and severe OA patients according to KL imaging diagnosis standards;
Detecting the expression level of miRNA-140in each group of joint fluid by a real-time fluorescent quantitative PCR method, and comparing the expression difference of miRNA-140in the joint fluid of normal and OA patients with different degrees by adopting one-way ANOVA variance analysis;
The correlation between the relative expression amount of miRNA-140in the synovial fluid and the KL classification was verified by the Spearman rank correlation test.
The invention also aims to provide a method for verifying the regulation and control effect of the miRNA-140 injected into the articular cavity on rat knee joint cartilage degeneration, which comprises the following steps: the OA model of the rat knee joint is constructed and identified.
Further, the specific method for verifying the regulation and control effect of the miRNA-140 injected into the joint cavity on rat knee joint cartilage degeneration comprises the following steps:
Taking SPF (specific pathogen free) SD (rat) rats which are mature in 3 months of age, taking 12 rats, wherein 3 rats are used as normal controls, the other 9 rats are used for establishing a rat knee joint OA (knee joint OA) model by a method of surgically removing medial meniscus and medial collateral ligament, 3 rats are killed at 4, 8 and 12 weeks after surgery, and the OA model is identified through general observation, tissue section staining (HE + toluidine blue) and Mankin's score;
Taking 42 rats, wherein 6 rats serve as normal controls and are not treated; the other 36 rats are randomly divided into an experimental group and a control group, each group comprises 18 rats, 5nmol/100 mu l of miRNA-140agomir is injected into the knee joint cavity of the rat in the experimental group through joint cavity puncture 1 week after the modeling, the control group is injected with miRNA-140control with the same dose, and the existence of complications after the injection of the joint cavities of the two groups of rats is observed;
Each group of 6 rats was sacrificed at 4, 8 and 12 weeks after modeling, and the cartilage degeneration degree of knee joints of the two groups of rats was compared by gross observation, histochemical staining (HE + toluidine blue) and Mankin's scoring method;
The regulation and control function and the molecular mechanism of the articular cavity injection miRNA-140 on the degeneration of the knee joint cartilage of the rats are verified by comparing the expression conditions of related proteins in the knee joint cartilage tissues of two groups of rats through immunohistochemical staining (Collagen II, MMP13 and ADAMTS-5) and protein expression average optical density values (MOD).
The invention successfully separates and cultures human normal and different OA chondrocytes in vitro, miRNA-140 is stably expressed in the normal chondrocytes, the expression level of the miRNA-140 is gradually reduced along with the increasing of the OA degree, the expression is the lowest in the severe OA chondrocytes, and the difference between groups has statistical significance (F is 56.32, and p is less than 0.05).
Along with the increase of the miRNA-140 micic concentration and the prolonging of the transfection time, the expression of the Collagen II gene and protein is gradually increased, and the expression of the MMP13 gene and protein is gradually reduced. At the transfection time of 72h at the concentration of 50nM, the differences in the expression levels of Collagen II and MMP13 genes and proteins were statistically significant compared to the control group (p < 0.05).
After 50nM miRNA-140mimic transfects chondrocytes for 72h, compared with a control group, the expression of Collagen II and Sox9 genes is increased, and the expression difference in normal, light and moderate OA chondrocytes has statistical significance (p is less than 0.05); and MMP13, ADAMTS-5 and Runx2 gene expression are reduced, wherein the difference of MMP13 expression in normal and mild OA chondrocytes and Runx2 expression in moderate OA chondrocytes is statistically significant (p < 0.05). In severe OA chondrocytes, the gene expression was not statistically different from that of the control group (p > 0.05). The expression change trend of the genes is opposite after miRNA-140inhibitor transfection.
after 50nM miRNA-140mimic transfection, 72h detection shows that compared with a control group, the average expression level of Collagen II protein in normal and various levels of OA chondrocytes is obviously increased, and the difference has statistical significance (p is less than 0.05); the expression levels of MMP13 and ADAMTS-5 proteins are reduced, wherein the expression difference of MMP13 in various levels of OA chondrocytes is statistically significant (p is less than 0.05), and the expression difference of ADAMTS-5 in normal, light and moderate OA chondrocytes is statistically significant (p is less than 0.05). The protein expression after miRNA-140inhibitor transfection has opposite trend.
miRNA-140 is stably expressed in human normal articular chondrocytes, and the expression level of the miRNA-140 is gradually reduced along with the increase of OA degree.
The optimal concentration of miRNA-140 micic transfected human articular chondrocytes is 50nM, and the optimal time for detecting the transfection effect is 72h after transfection.
The up-regulation of miRNA-140 expression in human articular chondrocytes can promote the expression of Collagen II and Sox9, and inhibit the expression of MMP13, ADAMTS-5 and Runx2, thereby playing a role in preventing and repairing the OA-like change of chondrocytes, and the role is more obvious in light and moderate OA chondrocytes.
miRNA-140 expression can be detected in normal and OA joint fluid, the expression of miRNA-140in OA joint fluid is obviously reduced compared with that in a normal group, and the difference between groups has statistical significance (F is 15.28, and p is less than 0.05).
The expression level of miRNA-140in synovial fluid gradually decreases with the increase of OA degree, and is significantly negatively correlated with KL grading (r is-0.92, p is less than 0.05).
The expression of miRNA-140 can be detected in normal and OA joint fluid of human, the expression level thereof is gradually reduced along with the increasing of OA degree, and the expression level is obviously and negatively correlated with the OA severity degree.
The OA model of the knee joint of the rat is successfully established, the average modeling operation time is 10.60 +/-1.80 min/mouse, the operation incisions are healed within 1 week after the operation, and the average Mankin's scores at the 4 th, 8 th and 12 th weeks after the operation are respectively 6.50 +/-1.52, 10.17 +/-0.75 and 12.83 +/-0.75.
After injecting miRNA-140agomir or miRNA-140control into the joint cavity, no complication such as joint infection or death of rats occurs.
At 4, 8 and 12 weeks after modeling, the degeneration degree of the articular cartilage of the rats in the experimental group is obviously lower than that of the rats in the control group through gross observation and histochemical staining, the Mankin's scores (3.50 +/-0.55, 6.33 +/-0.82 and 8.67 +/-1.21) of the articular cartilage tissues of the rats in the experimental group at each time point are lower than those of the rats in the control group (6.83 +/-1.17, 10.50 +/-1.05 and 13.17 +/-0.75), and the difference is statistically different (p < 0.05).
The Collagen II expression decreased gradually as the modeling time increased, while MMP13 and ADAMTS-5 expression increased gradually. At 4, 8 and 12 weeks after modeling, the Collagen II positive expression intensity in the articular cartilage tissue of the rats in the experimental group is higher than that in the control group, and the MMP13 and ADAMTS-5 positive expression intensity are lower than that in the control group. The Collagen II immunohistochemical MOD values at each time point of the experimental group were 11.03 + -1.54, 7.88 + -1.66 and 4.83 + -1.26 (x 10-3), respectively, and the differences between the control groups were 7.60 + -1.21, 4.45 + -1.35 and 1.47 + -0.31 (x 10-3), respectively, and were statistically significant (p < 0.05). The MMP13 immunohistochemical MOD values of 2.93 +/-0.51, 5.05 +/-0.98 and 7.92 +/-1.52 (multiplied by 10-3) at each time point of the experimental group, and the differences among the control groups are respectively 5.63 +/-1.07, 8.43 +/-1.09 and 11.70 +/-1.56 (multiplied by 10-3), and have statistical significance (p is less than 0.05). ADAMTS-5 immunohistochemical MOD values at each time point in the experimental group were 2.60. + -. 0.62, 3.70. + -. 1.05 and 8.85. + -. 1.73 (. times.10-3), and differences between the groups were statistically significant (p <0.05) for the control groups at 4.13. + -. 0.75, 9.72. + -. 2.04 and 18.17. + -. 5.20 (. times.10-3).
The OA model of the rat is successfully established, and the miRNA-140agomir injected into the joint cavity is safe and feasible.
The joint cavity injection of miRNA-140agomir can obviously slow down the degeneration process of rat articular cartilage.
The miRNA-140agomir injected into the joint cavity can obviously slow down the loss of Collagen II protein in the articular cartilage tissue, obviously inhibit the expression of MMP13 and ADAMTS-5 protein, and play an obvious role in delaying the degeneration of the articular cartilage of rats.
Drawings
FIG. 1 is a flow chart of a method for verifying the regulatory effect of miRNA-140in osteoarthritic chondrocytes, provided by the embodiments of the present invention.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The application of the principles of the present invention will be further described with reference to the accompanying drawings and specific embodiments.
A medicine for regulating the degeneration of articular cartilage is miRNA-140.
as shown in fig. 1: the invention provides a method for verifying the regulation and control effect of miRNA-140in osteoarthritis chondrocytes, which comprises the following steps:
S101: firstly, verifying the expression rule of miRNA-140in human normal and Osteoarthritis (OA) chondrocytes;
S102: secondly, screening the optimal condition of the miRNA-140 for transfecting the human chondrocytes;
S103: finally, the regulation and control function and mechanism of the miRNA-140 expression up-regulation or down-regulation on human OA chondrocytes are verified.
Further, the method for verifying the regulation and control effect of miRNA-140in osteoarthritis chondrocytes specifically comprises the following steps:
(1) According to the Classification standard of Kellgren and Lawrence (KL) OA imaging science, collecting human normal (KL 0 grade), light (KL1-2 grade), medium (KL 3 grade) and heavy (KL 4 grade) OA fresh cartilage specimens, and separating, culturing and identifying chondrocytes in vitro;
(2) Detecting the expression level of miRNA-140in normal and OA chondrocytes by adopting a fluorescent quantitative PCR method, and analyzing the expression change rule of the miRNA-140;
(3) Transfecting chondrocytes by miRNA-140mimic with different concentrations (0, 25, 50, 100 and 200nM), detecting the expression levels of cells Collagen II and MMP13 genes and proteins by PCR and Western blotting methods at different time points (24, 48 and 72h) after transfection, and screening the optimal concentration and the optimal time for detecting the effect of the miRNA-140mimic transfected chondrocytes;
(4) The miRNA-140 micic or miRNA-140inhibitor with the optimal concentration is adopted to transfect normal and OA chondrocytes, the expression levels of genes and proteins of cells Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 are detected at the optimal time, the expression differences of the genes and the proteins in various groups of chondrocytes after the miRNA-140 is up-regulated or down-regulated are compared, and the regulation and control effect and the molecular mechanism of the miRNA-140 on the OA chondrocytes are analyzed.
The invention provides a method for verifying the regulation and control effect of miRNA-140in osteoarthritis joint fluid, which comprises the following steps:
Detecting the expression rule of miRNA-140in human normal and OA joint fluid, and verifying the correlation between the expression level of miRNA-140 and the severity of OA.
Further, the method for verifying the regulation and control effect of the miRNA-140in osteoarthritis joint fluid specifically comprises the following steps:
Collecting 10 cases of knee joint fluid specimens of normal, light, medium and severe OA patients according to KL imaging diagnosis standards;
Detecting the expression level of miRNA-140in each group of joint fluid by a real-time fluorescent quantitative PCR method, and comparing the expression difference of miRNA-140in the joint fluid of normal and OA patients with different degrees by adopting one-way ANOVA variance analysis;
the correlation between the relative expression amount of miRNA-140in the synovial fluid and the KL classification was verified by the Spearman rank correlation test.
The invention provides a method for verifying the regulation and control effect of articular cavity injection miRNA-140 on rat knee joint cartilage degeneration, which comprises the following steps: the OA model of the rat knee joint is constructed and identified.
Further, the specific method for verifying the regulation and control effect of the miRNA-140 injected into the joint cavity on rat knee joint cartilage degeneration comprises the following steps:
Taking SPF (specific pathogen free) SD (rat) rats which are mature in 3 months of age, taking 12 rats, wherein 3 rats are used as normal controls, the other 9 rats are used for establishing a rat knee joint OA (knee joint OA) model by a method of surgically removing medial meniscus and medial collateral ligament, 3 rats are killed at 4, 8 and 12 weeks after surgery, and the OA model is identified through general observation, tissue section staining (HE + toluidine blue) and Mankin's score;
Taking 42 rats, wherein 6 rats serve as normal controls and are not treated; the other 36 rats are randomly divided into an experimental group and a control group, each group comprises 18 rats, 5nmol/100 mu l of miRNA-140agomir is injected into the knee joint cavity of the rat in the experimental group through joint cavity puncture 1 week after the modeling, the control group is injected with miRNA-140control with the same dose, and the existence of complications after the injection of the joint cavities of the two groups of rats is observed;
Each group of 6 rats was sacrificed at 4, 8 and 12 weeks after modeling, and the cartilage degeneration degree of knee joints of the two groups of rats was compared by gross observation, histochemical staining (HE + toluidine blue) and Mankin's scoring method;
The regulation and control function and the molecular mechanism of the articular cavity injection miRNA-140 on the degeneration of the knee joint cartilage of the rats are verified by comparing the expression conditions of related proteins in the knee joint cartilage tissues of two groups of rats through immunohistochemical staining (Collagen II, MMP13 and ADAMTS-5) and protein expression average optical density values (MOD).
The invention successfully separates and cultures human normal and different OA chondrocytes in vitro, miRNA-140 is stably expressed in the normal chondrocytes, the expression level of the miRNA-140 is gradually reduced along with the increasing of the OA degree, the expression is the lowest in the severe OA chondrocytes, and the difference between groups has statistical significance (F is 56.32, and p is less than 0.05).
Along with the increase of the miRNA-140 micic concentration and the prolonging of the transfection time, the expression of the Collagen II gene and protein is gradually increased, and the expression of the MMP13 gene and protein is gradually reduced. At the transfection time of 72h at the concentration of 50nM, the differences in the expression levels of Collagen II and MMP13 genes and proteins were statistically significant compared to the control group (p < 0.05).
after 50nM miRNA-140mimic transfects chondrocytes for 72h, compared with a control group, the expression of Collagen II and Sox9 genes is increased, and the expression difference in normal, light and moderate OA chondrocytes has statistical significance (p is less than 0.05); and MMP13, ADAMTS-5 and Runx2 gene expression are reduced, wherein the difference of MMP13 expression in normal and mild OA chondrocytes and Runx2 expression in moderate OA chondrocytes is statistically significant (p < 0.05). In severe OA chondrocytes, the gene expression was not statistically different from that of the control group (p > 0.05). The expression change trend of the genes is opposite after miRNA-140inhibitor transfection.
After 50nM miRNA-140mimic transfection, 72h detection shows that compared with a control group, the average expression level of Collagen II protein in normal and various levels of OA chondrocytes is obviously increased, and the difference has statistical significance (p is less than 0.05); the expression levels of MMP13 and ADAMTS-5 proteins are reduced, wherein the expression difference of MMP13 in various levels of OA chondrocytes is statistically significant (p is less than 0.05), and the expression difference of ADAMTS-5 in normal, light and moderate OA chondrocytes is statistically significant (p is less than 0.05). The protein expression after miRNA-140inhibitor transfection has opposite trend.
miRNA-140 is stably expressed in human normal articular chondrocytes, and the expression level of the miRNA-140 is gradually reduced along with the increase of OA degree.
The optimal concentration of miRNA-140 micic transfected human articular chondrocytes is 50nM, and the optimal time for detecting the transfection effect is 72h after transfection.
The up-regulation of miRNA-140 expression in human articular chondrocytes can promote the expression of Collagen II and Sox9, and inhibit the expression of MMP13, ADAMTS-5 and Runx2, thereby playing a role in preventing and repairing the OA-like change of chondrocytes, and the role is more obvious in light and moderate OA chondrocytes.
miRNA-140 expression can be detected in normal and OA joint fluid, the expression of miRNA-140in OA joint fluid is obviously reduced compared with that in a normal group, and the difference between groups has statistical significance (F is 15.28, and p is less than 0.05).
The expression level of miRNA-140in synovial fluid gradually decreases with the increase of OA degree, and is significantly negatively correlated with KL grading (r is-0.92, p is less than 0.05).
The expression of miRNA-140 can be detected in normal and OA joint fluid of human, the expression level thereof is gradually reduced along with the increasing of OA degree, and the expression level is obviously and negatively correlated with the OA severity degree.
The OA model of the knee joint of the rat is successfully established, the average modeling operation time is 10.60 +/-1.80 min/mouse, the operation incisions are healed within 1 week after the operation, and the average Mankin's scores at the 4 th, 8 th and 12 th weeks after the operation are respectively 6.50 +/-1.52, 10.17 +/-0.75 and 12.83 +/-0.75.
After injecting miRNA-140agomir or miRNA-140control into the joint cavity, no complication such as joint infection or death of rats occurs.
At 4, 8 and 12 weeks after modeling, the degeneration degree of the articular cartilage of the rats in the experimental group is obviously lower than that of the rats in the control group through gross observation and histochemical staining, the Mankin's scores (3.50 +/-0.55, 6.33 +/-0.82 and 8.67 +/-1.21) of the articular cartilage tissues of the rats in the experimental group at each time point are lower than those of the rats in the control group (6.83 +/-1.17, 10.50 +/-1.05 and 13.17 +/-0.75), and the difference is statistically different (p < 0.05).
The Collagen II expression decreased gradually as the modeling time increased, while MMP13 and ADAMTS-5 expression increased gradually. At 4, 8 and 12 weeks after modeling, the Collagen II positive expression intensity in the articular cartilage tissue of the rats in the experimental group is higher than that in the control group, and the MMP13 and ADAMTS-5 positive expression intensity are lower than that in the control group. The Collagen II immunohistochemical MOD values at each time point of the experimental group were 11.03 + -1.54, 7.88 + -1.66 and 4.83 + -1.26 (x 10-3), respectively, and the differences between the control groups were 7.60 + -1.21, 4.45 + -1.35 and 1.47 + -0.31 (x 10-3), respectively, and were statistically significant (p < 0.05). The MMP13 immunohistochemical MOD values of 2.93 +/-0.51, 5.05 +/-0.98 and 7.92 +/-1.52 (multiplied by 10-3) at each time point of the experimental group, and the differences among the control groups are respectively 5.63 +/-1.07, 8.43 +/-1.09 and 11.70 +/-1.56 (multiplied by 10-3), and have statistical significance (p is less than 0.05). ADAMTS-5 immunohistochemical MOD values at each time point in the experimental group were 2.60. + -. 0.62, 3.70. + -. 1.05 and 8.85. + -. 1.73 (. times.10-3), and differences between the groups were statistically significant (p <0.05) for the control groups at 4.13. + -. 0.75, 9.72. + -. 2.04 and 18.17. + -. 5.20 (. times.10-3).
The OA model of the rat is successfully established, and the miRNA-140agomir injected into the joint cavity is safe and feasible.
The joint cavity injection of miRNA-140agomir can obviously slow down the degeneration process of rat articular cartilage.
The miRNA-140agomir injected into the joint cavity can obviously slow down the loss of Collagen II protein in the articular cartilage tissue, obviously inhibit the expression of MMP13 and ADAMTS-5 protein, and play an obvious role in delaying the degeneration of the articular cartilage of rats.
The application of the principles of the present invention will be further described with reference to the following experiments and specific examples.
Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction, subchondral bone changes, periarticular osteophyte formation, and synovial inflammatory changes, and is commonly found in middle-aged and elderly patients with heavy-weight and active joints (e.g., knee, hip, spine, ankle, hand, etc.). Along with the progress of OA disease course, symptoms such as arthralgia, deformity and movement disorder can gradually appear, and the disability rate can reach 53 percent. The population base of China is large, the people are already in the aging society, the number of the OA patients is increased, but the specific pathogenesis of the OA is still unclear at present. Chondrocytes (chondrocytes) are the only cellular component of articular cartilage tissue, and synthesize a variety of Extracellular matrix (ECM) components including collagen, proteoglycans, and non-collagenous proteins. In normal articular cartilage, chondrocyte anabolism and catabolism are in a dynamic balance, which plays an important role in maintaining normal homeostasis and function of articular cartilage and is regulated by various factors. When this state of balance is disrupted or the associated regulatory system is dysfunctional, an imbalance in chondrocyte metabolism will promote chondrocyte terminal differentiation and ECM degradation, which if not repaired or prevented in time, will eventually lead to OA-like changes in articular cartilage.
microRNA (miRNA) is a highly conserved small non-coding RNA, is about 22 nucleotides long, can be combined with the 3' Untranslated regions (UTRs) of a target gene (mRNA) in a cytoplasm in a complete or partial complementary form to cause the degradation or the translation inhibition of the target mRNA, regulates the translation of the mRNA of about 1/3 of mammals, and plays an important role in the aspects of cell proliferation, differentiation, migration, aging, apoptosis and the like. In recent years, the research finds that miRNA-140 has cartilage specificity, is stably expressed in human normal articular cartilage, and plays a specific regulation role in the homeostasis and the function of the articular cartilage. At present, the expression change rule of miRNA-140in OA cartilage is still controversial, and the research finds that the expression of miRNA-140in OA cartilage tissues or cells is reduced compared with that of normal cartilage tissues or cells, but the research reports that the expression of miRNA-140in OA cartilage is increased compared with that of normal cartilage.
Therefore, in order to further explore the expression rule and regulation and control effect of miRNA-140in OA chondrocytes, the experiments in the part firstly detect the expression level of miRNA-140in human normal and OA chondrocytes, secondly screen the optimal condition of miRNA-140 micic transfection on human chondrocytes, and finally explore the regulation and control effect and molecular mechanism of miRNA-140 expression up-regulation or down-regulation on human normal and OA chondrocytes.
2 materials and methods
2.1 materials of the experiment
2.1.1 human fresh cartilage specimens
OA was classified on a scale of 0-4 according to the diagnostic criteria of Kellgren and Lawrence (KL) imaging (Table 1). A patient who performs above knee amputation or Total knee replacement (TKA) is selected from orthopedics department and emergency department of western medicine hospital of Sichuan university, and 5-10 cases of femoral condyle cartilage specimens of normal (KL 0 grade), mild OA (KL1-2 grade), moderate OA (KL 3 grade) and severe OA (KL 4 grade) are collected during operation. All specimen collections were approved by the patient and/or his family and signed with an informed consent.
TABLE 1 osteoarthritis Kellgren-Lawrence (KL) X-ray grading Standard
2.1.2 Primary reagents
Other reagents are domestic or imported analytical pure-grade high-quality reagents.
2.1.3 preparation of the Main solution
1) 0.2% collagenase solution: 20mg collagenase powder +10ml DMEM (high sugar) medium containing 5% FBS, prepared and then sterilized by filtration using a sterile disposable filter (pore size 0.2 μm) and prepared just before use.
2) 20% FBS medium: 20ml of FBS +80ml of DMEM (high-sugar) culture medium, uniformly mixing, preparing 100ml each time, subpackaging, storing at 4 ℃, and preparing after use.
3) 1% toluidine blue staining solution: after dissolving toluidine blue powder 0.2g +2ml 70% ethanol sufficiently, 18ml 1% NaCl solution (1g NaCl +100ml distilled water, ready to use) was added, and mixed well, and filtered with filter paper.
2.1.4 Main instruments, Equipment
Other instruments and equipment are all made in China or imported in recent years and are provided by science and technology park of western hospitals of Sichuan university.
2.1.5 Experimental consumables
The cell culture bottles/dishes, centrifuge tubes, pipette tips, cell sieves, filter screens and other experimental consumables with various specifications are all domestic or imported high-quality products.
2.2 Experimental methods
2.2.1 chondrocyte in vitro culture and identification
2.2.1.1 cartilage tissue specimen Collection
During the period from 6 months 2014 to 8 months 2015, fresh cartilage tissue specimens are collected in the western hospital of Sichuan university, and the whole process is strictly operated aseptically, and the specific method comprises the following steps:
1) after exposing the femoral condyle in the TKA operation, the femoral condyle cartilage is sliced and removed by a scalpel. For above knee amputation patients, after the limbs are cut off, the knee joint area is disinfected and paved with a sterile towel, and the cartilage tissue is cut after the femoral condyle is exposed. During the material drawing process, disinfectant or other irritant liquid is prevented from contacting cartilage of the material drawing area.
2) The cartilage pieces were placed in a clean sterile tray, washed three times with Normal Saline (NS), transferred to a 50ml sterile centrifuge tube containing DMEM (high sugar) medium, and transported to the laboratory for cell separation as soon as possible at low temperature.
2.2.1.2 isolation and culture of Primary chondrocytes
separating primary chondrocytes by adopting a trypsin + collagenase digestion method, which comprises the following steps:
1) Taking a proper amount of cartilage tissue samples, putting the cartilage tissue samples into a 50ml sterile centrifuge tube, and adding a small amount of culture medium until the samples are flooded. Cutting cartilage pieces into pieces of size <1mm3 with sterile long-handled tissue scissors, washing with sterile NS for 2 times, centrifuging, pouring out the NS, and drying.
2) Adding 0.25% trypsin solution with volume more than 3 times, shaking, digesting in water bath at 37 deg.C for 15-30min, centrifuging (1200r/min, 3min), sucking supernatant, and washing with sterile NS for 2 times.
3) Adding more than 3 times of 2% collagenase solution, shaking, and digesting in 37 deg.C water bath for more than 4 hr until the tissue mass substantially disappears and the digestive juice turns turbid.
4) Filtering with 200 mesh sterile stainless steel cell sieve, collecting filtrate, centrifuging (1500r/min, 10min), removing supernatant, suspending the precipitated cells with sterile NS, and centrifuging for 1 time.
Suspending and precipitating cells with 20% FBS culture medium, inoculating into culture bottle or culture dish according to cell density (0.5-2). times.105 cells/ml, culturing in 5% CO2 culture box at 37 deg.C, changing culture solution 1 time every 2-3 days, and digesting with trypsin solution when the cells grow over the bottom wall of the culture bottle/dish.
2.2.1.3 chondrocyte passage
1) When the chondrocytes proliferate to cover more than 80% of the bottom of the culture flask, the old culture medium is aspirated off, and PBS is added for washing for 2-3 times.
2) Adding a proper amount of 0.25% trypsin solution according to the size of a culture bottle/dish, slightly shaking to enable the digestive juice to flow over the surfaces of all cells, digesting for 1-3 minutes in an incubator, observing under a microscope, and adding an equal volume of 20% FBS culture medium to stop digestion when most of cells contract cytoplasm, increase gaps and round and float; the medium in the bottle/dish was aspirated by a pipette, the bottle/dish wall cells were repeatedly and sequentially blown, the cell suspension was collected and transferred to a 15ml centrifuge tube, and centrifugation was performed (1500r/min, 3-5 min).
3) discarding the supernatant, adding a culture medium containing 20% FBS to resuspend the cells, inoculating, placing in an incubator to continue culturing, observing and recording the growth condition of the chondrocytes under a microscope, and changing the culture solution 1 time every 2-3 days.
2.2.1.4 chondrocytes cryopreserving and resuscitating
The cell freezing and thawing needs to be performed slowly and quickly, and the specific method comprises the following steps:
1) Changing the solution regularly 1 day before freezing, and preparing cell preservation solution by using dimethyl sulfoxide (DMSO) and FBS (FBS) and a culture medium according to the ratio of 1:2:7 before digesting the cells.
2) Old culture medium was aspirated, washed 2-3 times with PBS, and cells were digested as described above.
3) Taking a proper amount of cell preservation solution to resuspend cell sediment, gently blowing and beating to uniformly distribute cells, counting by a cell counting plate, adjusting the final concentration of the cells in the preservation solution to be (5-10) multiplied by 106/ml, respectively filling the cell suspension into 1.5ml sterile cryopreservation tubes, screwing a sealing tube port, noting relevant information, putting the sterile cryopreservation tubes into a cell cryopreservation box, storing the sterile cryopreservation tubes in a refrigerator at minus 80 ℃, and transferring the cryopreservation tubes to a refrigerator at minus 150 ℃ for storing after 1-3 days.
4) During recovery, the cell freezing tube is taken out from a refrigerator at the temperature of-150 ℃, and is immediately put into a water bath kettle at the temperature of 37 ℃ under the condition of ensuring the sealing of the tube opening, and the content is melted as soon as possible by shaking.
5) Quickly sucking the cell suspension into a 50ml centrifuge tube, dropwise adding culture medium with more than 10 times of volume, gently mixing, centrifuging at low speed (1000r/min for 3min), removing supernatant, and washing with culture solution for 2 times.
6) Suspending the precipitated cells by using a 20% FBS culture medium, counting, inoculating, continuously culturing in an incubator, replacing the culture solution the next day, and then, replacing the culture solution conventionally and observing.
2.2.1.5 chondrocyte identification
The chondrocyte identification adopts methods such as observation under a light microscope, toluidine blue staining, type II collagen immunofluorescence staining and the like, and comprises the following steps:
(1) And observing the shape, density, adherence, growth and the like of the chondrocytes cultured in vitro under a microscope.
(2) Toluidine blue staining
1) and (3) placing the sterilized blood cover plate into a six-hole plate, inoculating chondrocytes with proper density, standing and culturing, observing the fusion rate of the chondrocytes on the blood cover plate under a microscope, and carrying out experiments on cell slide when the cell fusion rate reaches about 80%.
2) Removing culture medium, washing with PBS for 3 times, adding 4% paraformaldehyde phosphoric acid buffer solution until the blood cover sheet is completely submerged, fixing at 4 deg.C for 45-60min, removing paraformaldehyde, and washing with double distilled water for 3 times.
3) Adding 1% toluidine blue staining solution, soaking cell slide completely, staining at room temperature for 1.5-2 hr, removing excess staining solution, washing with double distilled water for 3-5 times, each for 3-5 min.
4) Dehydrating with 70% → 80% → 90% → absolute ethanol for 1min each.
5) Observing the dyeing condition under a microscope, repeating the previous two steps if necessary, airing after the dyeing is satisfied, sealing the piece by using neutral gum, and observing and acquiring the picture under the microscope.
(3) Type II collagen immunofluorescent staining
1) The cell slide was prepared and fixed as described above.
2) Adding 0.5% TritonX-100, incubating at room temperature for 10-20min, washing with PBS for 3 times, each for 3-5 min.
3) 1% BSA was prepared, wet-packed, blocked at 37 ℃ for 30min, the blocking solution was discarded, rabbit-derived polyclonal Collagen II antibody (1:100) was added, 40. mu.l of each slide was placed in the wet-packed chamber, and left overnight at 4 ℃.
4) PBS washing 3 times, each time for 5min, adding Alexa Fluor R488F (ab') 2fragment of coat anti-rabbitIgG (H + L) secondary antibody (1:200), each climbing sheet 40 μ L, placing in a wet box, and keeping away from light at 37 ℃ for 1H.
5) PBS was washed 3 times for 5min each, and 1. mu.g/ml DAPI stain was added and incubated at room temperature for 5 min.
6) Washing with PBS for 3 times, each for 5min, sealing with neutral gum, observing under fluorescent microscope, and collecting image.
2.2.2 detection of miRNA-140in Normal and OA chondrocytes
2.2.2.1 extraction of Total miRNA from cells
The specific method is operated according to the specification of the microRNA Purification Kit and comprises the following steps:
1) Culturing chondrocytes in a six-hole plate until the fusion rate reaches more than 80%, removing a culture medium, washing with PBS for 3 times, sucking up, adding 300 mu l of Buffer RL into each hole, shaking gently, tapping the hole plate, cracking for 5min, transferring a lysate into a 1.5ml centrifugal tube without RNAase pollution, adding 150 mu l of 96-100% ethanol into each tube, and uniformly mixing on a vortex instrument for 10 s.
2) assembling Collection Tube and Large RNA Removal Column, transferring the mixed solution to Column, centrifuging (14000g, 1min) to ensure that the mixed solution completely passes through Column, transferring the filtrate to a 1.5ml centrifugal Tube without RNAase pollution, adding 350 μ l of 96-100% ethanol, and mixing on a vortex apparatus for 10 s.
3) assembling the Collection Tube with the microRNA Enrichment Column, then transferring half of the mixed solution to the Column, centrifuging (>3500g, 1min) to ensure that the mixed solution completely passes through the Column, discarding the filtrate, and reassembling the Column and the Collection Tube. This step was repeated to complete the collection of small RNAs.
4) Add 400. mu.l of Wash Solution A to the microRNA Enrichment Column, centrifuge (14000g, 1min), ensure that the Wash Solution A all passes through the Column, discard the filtrate, reassemble the Column and the Collection Tube, repeat 2 times.
5) And (3) cleaning the microRNA Enrichment Column, ensuring that the Wash Solution A completely passes through the Column, discarding the filtrate, and reassembling the Column and the Collection Tube. The gel Column was again centrifuged for 2min to completely dry, the Collection Tube was discarded, and the microRNA Enrichment Column was placed in a supporting 1.7ml of the precipitation Tube.
6) add 50. mu.l of Elution Solution A to Column and centrifuge (200g, 2min) to ensure that all of Elution Solution A passes through Column and collect the eluate.
7) the previous two steps are repeated as necessary to ensure that the microRNA is collected to the maximum extent, and the collected microRNA sample is stored in a refrigerator at-70 ℃ and taken out when used.
2.2.2.2 determination of Total miRNA concentration in cells and integrity detection
1) And taking 2 mul of total miRNA sample, and detecting the concentration by an enzyme-labeling instrument.
2) The ratio of OD260/OD280 of the total miRNA is in the range of 1.8-2.2 ng/mul, the detection is carried out for 3 times, and the average value is taken.
3) Preparing 3% agarose gel: adding 30ml of TAE ((Tris-acetic acid, 0.04M Tris-acetic acid, 0.001M EDTA) solution into 0.3g of agarose powder, sealing with tin foil paper, then heating to boil in a microwave oven, repeating the operation until all the solution is dissolved and clarified, adding 2 mu l of Golden well stock solution, and uniformly mixing.
4) Pouring the gel into a proper mould, inserting a comb with a proper pore diameter, cooling for 30min, pulling out the comb, transferring the gel into an electrophoresis tank, and pouring 0.5 xTBE electrophoresis buffer solution to ensure that the liquid level is about 1-2mm higher than the gel level.
5) pipette 2. mu.l of 6 × Laoding buffer, 2. mu.l total microRNA, make up to 12. mu.l with RNAase-free water, mix well, microcentrifuge for 5s, sample, load 10. mu.l per well.
6) the negative end (black) was placed in the hole, voltage 150v, and electrophoresis was carried out for 20 min.
7) After electrophoresis is finished, the electrophoresis strips are observed under handheld ultraviolet light, 28S, 18S and 5S strips are visible from the hole end, and the brightness of the 28S strip is about 2 times that of the 18S strip.
2.2.2.4 reverse transcription of RT
And carrying out reverse transcription on the extracted total miRNA into cDNA by using a random primer and reverse transcriptase. Amplification was performed on a PCR instrument using the reverse AidMFrist Strand cDNA Synthesis Kit from Fermentas under the following conditions:
1) The following solutions were pre-mixed on an ice box:
2) Instantaneous centrifugation, pretreatment at 70 ℃ for 5min, and cooling on ice.
3) Then adding in sequence:
4) Performing instantaneous centrifugation, setting conditions by using a PCR instrument, and performing reverse transcription under the following conditions:
5) After reverse transcription, the cDNA was stored in a refrigerator at-20 ℃ for fluorescent quantitative PCR detection.
2.2.2.5 fluorescent quantitative PCR detection
1) Preparing a qPCR reaction system:
2) The qPCR reaction conditions were as follows:
3) The reaction system is carried out on a fluorescent quantitative PCR instrument, and when the amplification reaction of 39 cycles of amplification is finished, an amplification kinetic curve is drawn to determine the amplification cycle number (Ct value).
4) Data statistics were performed using a relatively quantitative method: the delta Ct1 is the average Ct value of the target gene in the experimental group-the average Ct value of the reference gene, the delta Ct2 is the average Ct value of the target gene in the control group-the average Ct value of the reference gene in the control group, the delta Ct is delta Ct 1-delta Ct2, and the relative expression amount of the target gene is 2-delta Ct.
2.2.2.6 gel electrophoresis of PCR detection products
The specific method is the same as the previous method.
2.2.3miRNA-140mimic/inhibitor transfection of human chondrocytes Condition exploration
According to the operation of the microRNA product use instruction provided by the Bo-Richardson Biotechnology company, transfection solutions are prepared before transfection, and strict aseptic operation is carried out.
2.2.3.1 preparation of transfection reagents
The operation is described according to the riboFECTTM CP transformation Kit, and the specific method is as follows:
1) Buffer (1X) was prepared by diluting Buffer (10X) with PBS water by flash centrifugation prior to use.
2) And taking out the Reagent, fully oscillating in a vortex oscillator, and standing at room temperature to restore the temperature to the room temperature for use.
2.2.3.2 preparing miRNA-140 micic transfection liquid with different concentrations
All manipulations strictly follow the RNA rules of operation, as follows:
1) Taking out 5nmol miRNA-140 lyophilized powder, centrifuging immediately before use, preparing 250 μ l (20nM) storage solution with sterilized double distilled water, subpackaging and storing in-80 deg.C refrigerator, taking out before use, and avoiding repeated freeze thawing.
2) miRNA-140 micid (v3) was diluted with 1 Xbuffer (v2), gently mixed, added with Reagent (v4), gently vortexed, without shaking, and incubated at room temperature for 0-15 min.
3) The mixture was added to the cell culture solution (v1) and gently mixed.
4) The amounts of reagents used during the preparation of the transfection solutions at different concentrations are shown in Table 2(6 well plate).
TABLE 2 preparation of miRNA-140mimic transfection solutions of different concentrations (6-well plate)
2.2.3.3miRNA-140mimic/inhibitor transfected chondrocytes
1) Appropriate amounts of mild OA chondrocytes were seeded into 6-well plates, and antibiotic-free medium was added per well to enable cell densities of 30-50% at transfection.
2) Removing old culture medium, adding 2ml of miRNA-140 imic transfection solution with different concentrations into the experimental group, replacing the culture medium of the control group normally, adding no transfection reagent, shaking up gently, and continuously culturing for 24-72h in the incubator.
2.2.3.4miRNA-140mimic/inhibitor transfection chondrocyte effect detection
2.2.3.4.1PCR detection of Collagen II and MMP13 gene expression level
1) Chondrocytes were cultured in 6-well plates, medium removed and washed 2 times with PBS.
2) 1ml of Trizol was added to each well, the lysed cells were repeatedly aspirated, and the cell lysate was transferred to a 1.5ml EP tube free from RNAase contamination and allowed to stand at room temperature for 5 min.
3) Add 200. mu.l of chloroform to each tube, seal, shake vigorously for 20s, stand at room temperature for 5min, and centrifuge (4 ℃, 12000g, 15 min).
4) The liquid in the tube was divided into three layers from top to bottom, the upper layer was total RNA liquid, the upper aqueous phase liquid was carefully aspirated and transferred to a new 1.5ml EP tube without RNAase enzyme contamination.
5) Adding equal volume of isopropanol, mixing, standing at room temperature for 10-15min, and centrifuging (4 deg.C, 12000g, 10 min).
6) Removing supernatant, adding 1ml of pre-cooled 75% ethanol, mixing, placing on ice box for 10min, centrifuging (4 deg.C, 12000g, 5min), removing supernatant, and drying at room temperature for 5-10 min.
7) Adding 15-30 μ l DEPC water, mixing, storing at-80 deg.C, and detecting total RNA concentration and subsequent detection.
8) The total RNA concentration determination, RNA integrity detection, reverse transcription, fluorescence quantitative PCR and PCR product gel electrophoresis method are the same as the previous method.
2.2.3.4.2Western blotting detection of expression level of Collagen II and MMP13 proteins
(1) Total protein extraction
1) Human chondrocytes were cultured in 6-well plates, the culture medium was removed, PBS was washed 2 times, PBS was aspirated off, and 1ml of lysis buffer (RIPA buffer: PMSF 100:1, 100mmol), collecting cells and lysate by cell scraping to a new 1.5ml ep tube, standing on an ice box for 1.5-2h, and fully lysing.
2) Centrifuging (4 deg.C, 12000rpm, 10-15min), carefully sucking the centrifuged supernatant, subpackaging and transferring to clean EP tube, adding 4 × Loading Buffer into a part of supernatant, boiling for 10min, and storing at-80 deg.C.
(2) BCA method for detecting protein concentration
1) Protein quantification was performed according to the BCA protein quantification kit protocol.
2) Setting the wavelength of the microplate reader to 562nm, placing the 96-well plate on the microplate reader for detection, making a standard curve, and then obtaining the protein concentration of the sample according to the light absorption value of the sample to be detected.
(3) SDS-PAGE electrophoresis
1) 12% of separation gel required by electrophoresis is prepared according to the following conditions, added into each group, mixed evenly and filled with gel.
12% separating glue formula
2) After 30-50min, pouring off absolute ethyl alcohol on the upper layer of the gel, fully sucking the gel by using absorbent paper, preparing the concentrated gel according to the following conditions, filling the concentrated gel between glass plates, immediately inserting a comb, slightly pulling out the comb after the concentrated gel is solidified, and washing gel holes by using distilled water.
Standard concentrated glue formula
3) The same amount of the protein tissue sample was filled to the same volume with Loading buffer and mixed well.
4) Adding sufficient electrophoresis buffer solution into the electrophoresis tank, adding the protein sample into each sample loading hole, after the sample loading is finished, covering the cover of the electrophoresis tank, switching on a power supply, setting the electrophoresis condition, adjusting the voltage to 80V, starting electrophoresis, and adjusting the voltage to 110V until the protein strips run to the separation gel.
5) The electrophoresis was terminated when bromophenol blue ran to the bottom of the plate.
(4) Rotary film
1) And soaking the PVDF membrane in methanol for 5min in advance, taking out the glass plate from the electrophoresis tank, prying the glass plate while flushing with running water, slightly scraping off the concentrated glue, and soaking the stripped glue in a membrane transferring solution.
2) The film transferring device is sequentially provided with a spongy cushion, filter paper, glue, an activated PVDF film, the filter paper and the spongy cushion, the glue needs to be aligned with the filter paper, the glue, the filter paper, the bubbles between the glue and the film are rolled by a glass rod, the film transferring device is closed, the film transferring device is placed into a transferring groove in the correct direction, a proper amount of ice grooves are added into the edge of the transferring groove, the current is constant at 400mA, and the film transferring operation is performed.
3) And (3) determining the membrane transferring time according to the size of the target protein, quickly taking out the membrane after the membrane transferring is finished, washing in a TBST buffer solution for 3min, and sealing with a sealing solution for 1 h.
(5) Immune response
1) The blotted PVDF membrane was blocked with 5% skim milk solution at room temperature for 1h or overnight at 4 ℃.
2) The primary antibody was diluted to the appropriate concentration, and after incubating the PVDF membrane for 1-2h at room temperature, it was washed 3 times with TBST on a decolourization shaker at room temperature, and once with TBS for 10 min.
3) the secondary HRP-labeled antibody was diluted with blocking solution in an appropriate ratio to give a secondary antibody working solution, and the membrane was placed therein and incubated at room temperature for 1 hour with shaking. Decoloration was performed with TBST at room temperature for 3 times 10min in a shaker, and TBS was performed for 10 min.
(6) Development and fixing reactions
After adding the color developing substrate, transferring the signal on the transfer printing film to an X-ray film according to an X-ray film developing method.
(7) Semi-quantitative analysis
The films were scanned and then analyzed for gray scale values of the target protein bands and internal controls using Image J software for semi-quantitative analysis.
2.2.4 Regulation and mechanism of miRNA-140 on OA chondrocytes
2.2.4.1 the method for culturing normal and OA chondrocytes is the same as before.
2.2.4.2miRNA-140mimic/inhibitor/control transfection of normal and OA chondrocytes
(1) Experimental groups each group of chondrocytes was divided into: a blank control group, a miRNA-140 micic transfection group, a miRNA-140inhibitor transfection group and a miRNA-140control transfection group.
(2) Transfection
the transfection concentration is the optimal transfection concentration screened in the early stage, the miRNA-140inhibitor transfection concentration is 2 times of the mimic concentration, the control transfection concentration is the same as the mimic concentration, a blank control group is used for replacing a culture medium normally and is not added with any transfection reagent, and the specific transfection method is the same as the previous method.
2.2.4.3 detection of transfection Effect
(1) PCR detection of Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 Gene expression
1) The specific method is the same as the previous method.
2) Design of related primers
(2) Western blotting detection of Collagen II, MMP13 and ADAMTS-5 protein expression
The specific method is the same as the previous method.
2.3 statistical analysis
Statistical analysis was performed on all data using SPSS 22.0 software, the data measured using mean + -SD, the two group comparisons using Mann-Whitney U test, and the multiple group comparisons using one-way ANOVA analysis of variance. Statistical differences were assigned a p < 0.05.
3 results
3.1 culture and identification of human articular chondrocytes
3.1.1 cartilage tissue specimen origin patient basic conditions
36 femoral condyle cartilage specimens of patients with normal (KL 0 grade), moderate (KL 3 grade) and severe (KL 4 grade) OA are collected from orthopedics department and emergency department of western medicine hospital of Sichuan university, and the basic data of the patients are shown in Table 3.
TABLE 3 basic data of patients from cartilage tissue specimens
3.1.2 chondrocyte morphology and staining identification
(1) morphological observation
When observed under an inverted microscope, the adherent human normal chondrocytes are elliptical and polygonal, the cell morphology changes along with the increase of OA degree, and the cells with long fusiform shapes increase and are similar to fibroblast-like cells.
(2) Toluidine blue staining
And (3) staining the chondrocyte slide with toluidine blue, and then observing the stained chondrocyte slide under an upright microscope, wherein the chondrocyte nucleus is stained with deep blue, the cytoplasm is stained with bluish purple, the staining is positive, and the toluidine blue staining characteristic of the chondrocyte is met.
(3) Collagen II immunofluorescent staining
the chondrocyte slide is taken to carry out Collagen II immunofluorescence staining and then is placed under an upright fluorescence microscope for observation, a large amount of green fluorescence can be seen in chondrocyte cytoplasm, and the characteristics of the Collagen II immunofluorescence staining of the chondrocyte are met.
3.2 expression change rule of miRNA-140in human OA chondrocyte
The relative expression level of miRNA-140in chondrocytes is shown in Table 4. miRNA-140 is stably expressed in normal articular chondrocytes, and is obviously reduced in OA chondrocytes compared with normal groups, and the difference between groups has statistical significance (F is 53.62, p is less than 0.001). As the OA degree increases, the expression of miRNA-140 gradually decreases, and the expression of miRNA-140in severe OA chondrocytes is lowest.
TABLE 4 relative expression levels of miRNA-140in human normal and OA chondrocytes
3.3 optimal transfection concentration and detection time for transfecting human articular chondrocyte by miRNA-140
3.3.1 optimal concentration of miRNA-140mimic transfected chondrocytes
Along with the increase of the miRNA-140 micic concentration, the expression of the Collagen II gene and protein is gradually increased, and the expression of the MMP13 gene and protein is gradually reduced. Compared with the blank control group, when the miRNA-140 micic concentration is 50nM, the differences of the Collagen II and MMP13 gene and protein expression are statistically significant (p is less than 0.05). When the miRNA-140 micic concentration is more than 50nM, the expression of the Collagen II gene and protein is further increased, while the expression of the MMP13 gene is further reduced, but compared with the 50nM group, the difference is more and less statistically significant. Therefore, 50nM was chosen as the optimal concentration for miRNA-140mimic transfection of human chondrocytes in this study.
3.3.2 optimal time for detecting effect of miRNA-140mimic transfection chondrocyte
In the blank control group, after chondrocytes are cultured for 72h, the gene and protein expression of Collagen II and MMP13 are increased compared with those in the 24h group. Compared with the group of 24h, after 72h of miRNA-140mimic transfection, the expression of Collagen II gene and protein is obviously increased, the expression of MMP13 gene and protein is obviously reduced, and the difference is statistically significant (p < 0.05). Therefore, 72h after transfection was selected as the optimal time for detecting the effect of miRNA-140mimic transfection on human chondrocytes in the study.
3.4 Regulation of OA chondrocyte-related gene and protein expression by up/down regulation of miRNA-140 expression
According to the screening result of the previous step, 50nM miRNA-140 micic or 100nM miRNA-140inhibitor is used for transfecting normal and OA chondrocytes, and the expression of genes and proteins of Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 in the cells is detected after 72 hours.
3.4.1 Regulation of expression Up/Down-Regulation of miRNA-140 on the expression of Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 genes in each group of chondrocytes, there was no significant difference in the expression of each gene in the miRNA-140control group compared with the blank control group (p > 0.05).
Compared with the miRNA-140control group, the expression of the genes Collagen II and Sox9 in the miRNA-140 mic group is increased, and the expression difference of the genes Collagen II and Sox9 in normal, light and moderate OA chondrocytes has statistical significance (p is less than 0.05); and MMP13, ADAMTS-5 and Runx2 gene expression are reduced, wherein the difference of MMP13 expression in normal and mild OA chondrocytes and Runx2 expression in moderate OA chondrocytes is statistically significant (p < 0.05). In severe OA chondrocytes, the expression of 5 genes in the miRNA-140mimic group is not statistically different from that in the miRNA-140control group (p > 0.05).
Compared with the miRNA-140control group, the miRNA-140inhibitor group Collagen II and Sox9 gene expression is reduced, wherein the expression difference of Collagen II in normal, mild and severe OA chondrocytes is statistically significant (p <0.05) (, the expression difference of Sox9 in normal and severe OA chondrocytes is statistically significant (p < 0.05); the MMP13, ADAMTS-5 and Runx2 gene expression are increased, wherein the expression difference of MMP13 and ADAMTS-5 in normal and moderate OA chondrocytes is statistically significant (p < 0.05); and the expression difference of Runx2 in normal, mild and moderate OA chondrocytes is statistically significant (p <0.05)
3.4.2 influence of up/down regulation of miRNA-140 expression on Collagen II, MMP13 and ADAMTS-5 protein expression
Compared with a blank control group, the expression of the Collagen II protein of the miRNA-140mimic group is obviously increased, and the expression difference in human normal and various levels of OA chondrocytes has statistical significance (p is less than 0.05); the MMP13 and ADAMTS-5 protein expression is obviously reduced, wherein the expression difference of MMP13 in various levels of OA chondrocytes is statistically significant (p is less than 0.05), and the expression difference of ADAMTS-5 in normal, mild and moderate OA chondrocytes is statistically significant (p is less than 0.05).
Compared with a blank control group, the expression of the Collagen II protein in the miRNA-140inhibitor group is reduced, and the expression difference in normal chondrocytes and chondrocytes at all levels has statistical significance (p is less than 0.05); the MMP13 and ADAMTS-5 protein expression were increased compared with the blank control group, and the expression difference in normal and various levels of chondrocytes was statistically significant (p < 0.05).
Discussion 4
Osteoarthritis (OA) is a common degenerative disease, the pathogenesis of which is not well-defined, the degeneration of articular cartilage or the capacity of chondrocytes to regenerate after terminal differentiation are very weak, and no effective treatment has been available so far. In normal articular chondrocytes, anabolism and catabolism are in a dynamic equilibrium state and are regulated by a variety of factors. Recently, microRNA-140 is discovered to have cartilage specificity, plays an important role in maintaining normal homeostasis and functions of articular cartilage, and simultaneously participates in the generation and development of OA. The expression of the OA group miRNA-140 is obviously reduced compared with that of a normal group in knee joint cartilage tissues at the earliest time, and further, the OA-like change is shown in miRNA-140 knockout mice at 1 month of age, while the OA-like change is not shown in transgenic mice over expressing miRNA-140, and even a certain resistance effect is shown to antigens for inducing OA. In contrast, Swinger and Wheeler et al have found that the expression of miRNA-140in OA cartilage is significantly higher than that in normal cartilage by comparing the expression change of miRNA-140in normal (femoral neck fracture) and OA (total hip arthroplasty) femoral head articular cartilage, which makes the expression change rule of miRNA-140in articular cartilage controversial. Firstly, the expression change conditions of miRNA-140in human normal and OA chondrocytes are detected, the used chondrocytes are from knee joint cartilage tissues, and the result shows that the expression quantity of miRNA-140 is gradually reduced along with the increase of OA degree, and the research results are consistent. The reason for the contradictory results that the expression rules of miRNA-140in cartilage tissues or cells from hip and knee joints are diametrically opposite is not clear, and the contradictory results are probably related to different cartilage specimen sources, and no report comparing the expression change trends of miRNA-140in different articular cartilages such as hip and knee is reported in the literature at present. Because the negative weight of the hip and the knee joint is large and the incidence rate of OA is relatively high, the comparative analysis of the expression change difference of the miRNA-140 among the articular cartilages at different parts can be tried in the future, and a new clue can be provided for the role of the miRNA-140in the pathogenesis of OA and the subsequent treatment research.
Articular cartilage degeneration is one of the main features of OA, and mainly involves degradation of ECM such as Collagen type ii (Collagen ii) and proteoglycans (agrrechans). MMP-13 and ADAMTS-5 are considered to be the most important hydrolases in cartilage matrix degrading enzymes at present, L and the like also find that miRNA-140 is a negative regulatory factor of MMP13, the expressions of miRNA-140 and MMP13 are increased in the process of inducing OA-like change of chondrocytes by using proinflammatory factor IL-1 beta, and miRNA-140 can inhibit MMP13 expression, and the inventors further find that miRNA-140 can be directly combined with UTR sites at the end of MMP 133' by adopting luciferase reporter gene analysis. After the expression rules of miRNA-140in normal and OA chondrocytes are determined, miRNA-140mimic or miRNA-140inhibitor transfection is further used for up-regulating or down-regulating expression of miRNA-140in the chondrocytes, and the result shows that the up-regulating expression of miRNA-140 can reduce MMP13 gene and protein expression in the cells, wherein the protein expression difference is more obvious, and the protein expression difference is consistent with the action mechanism of miRNA-140, namely the protein expression is regulated and controlled at the post-transcriptional level after being combined with target mRNA. It was further demonstrated that miRNA-140 can prevent and repair chondrocyte OA-like changes by inhibiting the expression of matrix degrading enzyme MMP 13.
Besides MMP13, miRNA-140 up-regulation was also found to inhibit ADAMTS-5 gene and protein expression, and although the decrease in gene expression was not statistically different from the control group, the decrease in protein expression was statistically different in normal and light and moderate OA chondrocytes from the control group, which further suggested that miRNA-140 mainly inhibits ADAMTS-5 expression at post-transcriptional level. M and the like find that the intervention of proinflammatory factor IL-1 beta can reduce the expression of miRNA-140 of chondrocytes and increase the expression of ADAMTS-5 in vitro experiments; when miRNA-140 expression is increased by gene transfection, the induction of IL-1 beta is obviously weakened. Further, it was found that ADAMTS-5 expression was increased in miRNA-140-/-mice and decreased in miRNA-140 transgenic mice, and in vitro luciferase reporter gene analysis also found that the UTR at the' end of ADAMTS-53 contained miRNA-140 binding sites. Therefore, inhibition of ADAMTS-5 expression is also one of the mechanisms by which miRNA-140 prevents articular cartilage degeneration.
Sox9 is considered as one of the main transcription factors for regulating chondrogenesis, is involved in regulating the differentiation process of Mesenchymal Stem Cells (MSCs) to cartilage cells, and can also regulate the expression of cartilage-specific proteins such as Collagen II, IX and XI, maintain the normal proliferation rate of chondrocytes and prevent the hypertrophic differentiation of chondrocytes. Sox9 expression in OA cartilage tissue is reduced compared with normal cartilage, MMP13 expression is increased after silencing Sox9 gene, and Sox9 can promote miRNA-140 expression by combining promoter region specific sequences, which is also proved in Sox9 gene knockout and transgenic mouse research. Therefore, Sox9 is currently widely regarded as an upstream regulatory factor of miRNA-140. However, it was found that up-regulation of miRNA-140 expression in chondrocytes also promoted increased Sox9 expression, and that the difference between normal and light and moderate OA chondrocytes was statistically significant compared to the control group; conversely, down-regulation of chondrocyte miRNA-140 expression also inhibited Sox9 expression, statistically significant compared to control differences in normal and severe OA chondrocytes. Whether this suggests that miRNA-140 as a downstream factor may also feedback regulate Sox9 expression? K and the like find that the miRNA-140 can reduce the expression level of Sox9 protein, and the expression of Sox9mRNA has no obvious change, which suggests that the miRNA-140 can up-regulate the expression of Sox9 at the post-transcriptional level. It has also been found that up-regulation of miRNA-140 gene expression can inhibit RALA (a small GTPase in the TGF- β pathway) expression, whereas RALA inhibition can cause a significant increase in Sox9 protein expression, but Sox9mRNA expression is not significantly affected. As described above, Sox9 expression was found to be reduced after miRNA-140 down-regulation, especially in normal and severe OA chondrocytes, which is similarly in conflict with the results of the K et al study. As the expression change of the Sox9 protein is not detected, the related detection is further perfected in subsequent experiments, and the phase regulation action and mechanism of miRNA-140 and Sox9 in chondrocytes are deeply explored. Furthermore, Sox9 inhibits Runx2 transcriptional activity, which induces hypertrophic differentiation of chondrocytes, as will be discussed in detail later.
Runx2 is one of the major transcription factors regulating bone formation, and is highly expressed in hypertrophic chondrocytes, but hardly expressed in normal articular chondrocytes. It has been demonstrated that Runx2 can promote MMP13, Collagen X and alkaline phosphatase (ALP) expression in combination with the corresponding gene promoter, thereby promoting chondrocyte terminal differentiation and endochondral osteogenesis [21 ]. By studying the action mechanism, Runx2 mainly acts by combining with Smad to form a Runx2-Smad complex: upon binding of Smad3 to Runx2, Runx2 transcriptional activity is inhibited [28 ]; while upon binding Smad1 to Runx2, Runx2 transcriptional activity is activated [29 ]. Smad1, Smad3 are key proteins in the TGF-beta/BMP signaling pathway Smads pathway [5], which also suggests that Runx2 has a regulatory effect on chondrocytes, at least in part, by regulating the TGF-beta/BMP signaling pathway. Tuddenham et al found that miRNA-140 could promote Runx2 expression by inhibiting histone deacetylase 4 (HDAC 4) in the study of long and flat bone formation. However, no changes in HDAC4 gene or protein expression were found by Miyaki et al in miRNA-140-/-rat growth plates or articular cartilage [15 ]. In the present study, the expression of Runx2 is reduced after miRNA-140 is up-regulated, and the difference is statistically significant in moderate OA chondrocytes compared with the difference of a control group, which is contrary to the conclusion that miRNA-140 can promote Runx2 expression in an osteogenesis process reported by Tuddenham. This also suggests that miRNA-140 may play different regulatory roles in the osteogenesis process and the cartilage degeneration process on Runx2, and the mutual regulation relationship between miRNA-140 and Runx2 in the articular cartilage degeneration process still needs to be further studied.
In summary, this section finds that: the expression of miRNA-140in human articular chondrocytes is gradually reduced along with the increase of OA degree, and the miRNA-140in OA chondrocytes is up-regulated through gene transfection, so that the expression of Collagen II and Sox9 can be promoted, and the expressions of MMP13, ADAMTS-5 and Runx2 are inhibited, so that the effect of preventing and repairing the OA-like change of chondrocytes is achieved, and the effect is more obvious in light and moderate OA chondrocytes. This suggests that miRNA-140 may be an important entry point for preventing and treating OA, but most studies on treating OA by miRNA-140 are in vitro chondrocyte experiments and gene knockout/transgenic animal experiments, which has limited practical guidance for exploring the treatment of OA by miRNA-140. It was found that injecting miRNA-210 into knee joint cavities of rats can promote repair of ACL and meniscus injury by increasing the expression of Vascular Endothelial Growth Factor (VEGF) and fibroblast growth factor 2(FGF 2). Therefore, the application of miRNA-140 to the articular cavity may provide a new research method and theoretical basis for OA gene therapy, but no related literature report exists at present, and the effect and mechanism of the application of miRNA-140 to the articular cavity in subsequent experiments on cartilage degeneration are further provided.
5 conclusion
(1) miRNA-140 is stably expressed in human normal articular chondrocytes, and the expression level of the miRNA-140 is gradually reduced along with the increase of OA degree.
(2) The optimal concentration of miRNA-140 micic transfected human articular chondrocytes is 50nM, and the optimal time for detecting the transfection effect is 72h after transfection.
(3) The up-regulation of miRNA-140 expression in human articular chondrocytes can promote the expression of Collagen II and Sox9, and inhibit the expression of MMP13, ADAMTS-5 and Runx2, thereby playing a role in preventing and repairing the OA-like change of chondrocytes, and the role is more obvious in light and moderate OA chondrocytes.
The present invention is further illustrated below in connection with the expression pattern of miRNA-140in osteoarthritic joint fluid.
1. Materials and methods
1.1 Experimental materials
1.1.1 human Knee Joint fluid
OA was graded according to KL imaging diagnostic criteria (same as the first part), and 10 cases of knee joint fluid specimens were collected from orthodox, mild, moderate and severe OA patients at orthopedic housing department and outpatient joint cavity puncture room of western hospital, university of sichuan, and joint fluid was collected from joint cavity drug-injected patients only before the first injection. All specimen collections were approved by the patient and/or his family and signed with an informed consent.
2.1.2 Primary reagents
Other reagents are domestic or imported analytical pure-grade high-quality reagents.
1.1.3 Main Instrument
Other instruments and equipment are all made in China or imported in recent years and are provided by science and technology park of western hospitals of Sichuan university.
1.1.4 Main Experimental consumables
Experimental consumables such as centrifuge tubes and gun heads with various specifications and without RNase pollution are all domestic or imported high-quality products.
1.2 Experimental methods
1.2.1 Knee Joint fluid Collection, Split charging and preservation
1) The synovial fluid was immediately transferred to a 15ml sterile RNAase-free centrifuge tube after extraction, and centrifuged (3000rpm, 5 min).
2) and taking the supernatant, subpackaging into 1ml of RNAase-free 1.5ml centrifugal tubes, and placing into a liquid nitrogen tank for storage, wherein the supernatant is taken out when in use.
1.2.2 Total miRNA extraction from synovial fluid
The method is operated according to the instruction of the microRNA Purification Kit, and comprises the following specific steps:
1) Mu.l of synovial fluid was transferred to a 1.5ml centrifuge tube without RNAase contamination, 250. mu.l of Buffer RL was added, and the mixture was vortexed and mixed for 15 seconds.
2) Add 150. mu.l 96-100% ethanol and mix well on a vortex apparatus for 10 s.
3) Collection Tube was assembled with Large RNA Removal Column, and the cell lysate and ethanol mixture was transferred to Column and centrifuged (14000g, 1min) to ensure that the mixture was all passed through Column, and flowthrough was retained.
4) Flowthrough was transferred to a 1.5ml centrifuge tube without RNAase contamination, 350 μ l 96-100% ethanol was added and mixed well on a vortexer for 10 s.
5) Assembling the Collection Tube with the microRNA Enrichment Column, then transferring half of the mixed liquor obtained in the previous step to the Column, centrifuging (>3500g, 1min) to ensure that the mixed liquor completely passes through the Column, discarding flowthrough, and reassembling the Column and the Collection Tube.
6) And repeating the previous step to finish the collection of the small RNA.
7) Add 400. mu.l of Wash Solution A to the microRNA Enrichment Column, centrifuge (14000g, 1min), after ensuring that Wash Solution A has completely passed through the Column, discard flowthrough, reassemble the Column and the Collection Tube.
8) Repeating the previous step for 2 times, cleaning the microRNA Enrichment Column, discarding flowthrough after ensuring that the Wash Solution A completely passes through the Column, and reassembling the Column and the Collection Tube.
9) The gel Column was again centrifuged for 2min to completely dry, the Collection Tube was discarded, and the microRNA Enrichment Tube was placed in a supporting 1.7ml of the precipitation Tube.
10) Add 50. mu.l of Elution Solution A to Column and centrifuge (200g, 2min) to ensure that all of Elution Solution A passes through Column and collect the eluate.
11) the first two steps are repeated as necessary to ensure maximal collection of micrornas.
12) And storing the collected microRNA sample in a refrigerator at-70 ℃ and taking out the sample when the sample is used.
1.2.3 determination of Total MicroRNA concentration and integrity of synovial fluid
the specific method is the same as the previous method.
1.2.4 reverse transcription RT
the specific method is the same as the previous method.
1.2.5 fluorescent quantitative PCR detection
The specific method is the same as the previous method.
1.2.6PCR detection product gel electrophoresis
The specific method is the same as the previous method.
1.3 statistical analysis
Statistical analysis is carried out by adopting SPSS 22.0 software, the relative expression quantity of miRNA-140 among multiple groups is compared among groups by adopting one-way ANOVA variance analysis, and meanwhile, the correlation between the relative expression quantity of miRNA-140 and OAKL grading is analyzed by adopting Spearman grade correlation rank and test. Statistical differences were considered to be p < 0.05.
2 results
2.1 basic conditions of patients from whom synovial fluid specimens are derived
10 cases of knee joint fluid specimens of normal (KL 0 grade), mild (KL1-2 grade), moderate (KL 3 grade) and severe (KL 4 grade) OA patients are collected from orthopedics department of residence and clinic joint cavity puncture room of Wagner university Hospital, Sichuan, the basic information of the patients is shown in Table 5, and each group of typical X-ray tablets are obtained.
TABLE 5 basic data of patients from synovial fluid specimens
2.2 rules of variation in expression of miRNA-140in human Knee joint fluid
miRNA-140 expression can be detected in normal and OA joint fluid, the expression of miRNA-140in OA joint fluid is obviously reduced compared with that in normal group, and the difference between groups has statistical significance (F is 15.275, p is less than 0.001). As OA levels increased, miRNA-140 expression decreased gradually, with minimal miRNA-140 expression in severe OA synovial fluid (Table 6).
TABLE 6 relative expression level of miRNA-140in human knee joint fluid
2.3 correlation analysis of miRNA-140 expression in human Knee synovial fluid with OA severity
The miRNA-140 relative expression level in human knee joint fluid is remarkably and negatively correlated with KL grading by using a Spearman grade correlation rank sum test analysis (r is-0.924 p < 0.001).
Discussion of 3
OA is usually caused by heavy and active joints, such as knee, hip, spine (cervical and lumbar), ankle and hand, and is usually caused by single or several joints [6 ]. The light and moderate OA mainly takes conservative treatment as main treatment, and the treatment modes comprise physical therapy, oral anti-inflammatory analgesics (NSAIDs) and cartilage nutritious drugs (glucosamine, chondroitin sulfate and the like), joint cavity injection sodium hyaluronate or steroid hormone and the like. Severe OA patients have significant joint pain and deformity, have a large impact on quality of life, have poor conservative treatment effects and are prone to relapse, and joint replacement becomes the first choice for a large proportion of patients in order to alleviate symptoms and improve quality of life [7 ]. Although the joint replacement surgery can finally relieve the pain of the affected joint and improve the function of the joint, the surgery has a plurality of complications, for example, the surgery has great physical and mental impact on the patient, a certain time of recovery period after the surgery, certain risks of infection, thrombophlebitis formation, prosthesis loosening and the like, the problem of artificial joint revision and the like also exists for the patient with a long life expectancy, and meanwhile, certain burden is caused to the patient economically. Therefore, finding new methods to effectively delay, prevent and even repair the degeneration of articular cartilage has been the focus and difficulty of OA-related research. As shown in the previous experiments, it has been found that miRNA-140 can be an important entry point for treating OA, but since OA is often single-shot or several joint diseases, the risk and effect of applying miRNA-140 systemically to treat OA are uncertain, and there is no report on applying miRNA systemically at present, so that it is probably more feasible to apply miRNA-140 locally.
Stably expressing miRNA-140in human articular cartilage, gradually decreasing the expression level with the increase of OA degree, and guessing whether miRNA-140 expression also exists in synovial fluid? The invention proves that the miRNA-140 is expressed in human knee joint fluid, and the expression quantity of the miRNA-140 is gradually reduced along with the increasing of the OA degree, and is obviously and negatively related to the OA severity degree, which is consistent with the expression change rule of the miRNA-140in articular chondrocytes. Aiming at the problem of the source of miRNA-140in synovial fluid, no relevant literature report is available at present. Chondrocytes are the only cellular component of cartilage tissue in which mature miRNA-140 is synthesized. In OA cartilage tissue, the number of chondrocytes is reduced, and the synthesis amount of miRNA-140 is reduced. Therefore, it is presumed that miRNA-140in the knee joint fluid may be derived from chondrocytes in the articular cartilage tissue, but the specific source and its mechanism of production remain to be further developed. Furthermore, is synovial fluid secreted from the articular inner synovial tissue and is able to penetrate into the cartilage matrix to provide the cartilage cells with nutritional support, and since it has been demonstrated that miRNA-140 is expressed in synovial fluid, is miRNA-140 able to act on cartilage cells by penetrating into cartilage matrix with synovial fluid? The literature is consulted to find that no relevant report exists at present.
Cartilage degeneration is mainly characterized by the terminal differentiation of chondrocytes and the degradation of ECM, while MMP-13 and ADAMTS-5 are the most important hydrolases of matrix degrading enzymes. The former part of experiments prove that the miRNA-140 expression can promote the expression of Collagen II in articular chondrocytes, inhibit the expression of MMP13 and ADAMTS-5, and play an obvious role in preventing and repairing the OA-like change of the chondrocytes. Based on the results of the experiments, we speculate whether the similar effect can be achieved by injecting exogenous miRNA-140 into the joint cavity to improve the expression level of miRNA-140in the joint fluid. The reference finds that no research report of the local application of the miRNA-140 joint cavity exists at present. S, et al, injected miRNA-210 into the knee joint cavity of a rat and found that the miRNA-210 applied to the joint cavity can significantly improve the expression of Vascular Endothelial Growth Factor (VEGF) and fibroblast growth factor 2(FGF2) in the ligament, promote angiogenesis and finally accelerate ACL repair [4 ]. Subsequently Kawanishi et al further found that rat knee joint cavity injection of miRNA-210 could promote repair of meniscal white zone injury by upregulating Collagen II, VEGF, and FGF2 expression in meniscal cells [5 ].
Therefore, assumptions are made based on prior results and prior literature support: the miRNA-140 can effectively delay, prevent and even repair the degeneration of the articular cartilage through injecting into the articular cavity. To validate this hypothesis, the rat knee OA model will be first constructed, then rat miRNA-140agomir is injected by joint cavity puncture, and finally the rat knee cartilage degeneration degree is observed and its action mechanism is explored by selecting an appropriate time point in the OA process, in the next step of validation.
4 conclusion
The expression of miRNA-140 can be detected in normal and OA joint fluid of human, the expression level thereof gradually decreases with the increasing of OA degree, and the expression level is obviously and negatively correlated with the OA severity degree.
The invention is further explained by combining the regulation and control function and mechanism of miRNA-140 injected into the articular cavity on the cartilage degeneration of the knee joint of the rat.
1 materials and methods
1.1 Experimental materials
1.1.1 Experimental animals
A total of 54 SPF male SD rats developed and matured at 3 months of age were purchased from Daoshuo technologies, Inc., and had a body weight of 300. + -.20 g. The rats are purchased and then raised in the experimental animal center of Sichuan university, the environmental temperature is maintained at 16-25 ℃, the relative humidity is maintained at 60-80%, the rats are fed with food and water conventionally, and the rats are raised in 4 cages for at least one week before the experiment. All animal manipulations were performed under the university of Sichuan laboratory animal Care regulations.
2.1.2 Primary reagents
other reagents are domestic or imported analytical pure-grade high-quality reagents.
1.1.3 preparation of the Main solution
0.5% toluidine blue staining solution: after dissolving toluidine blue powder 0.1g +2ml 70% ethanol sufficiently, 18ml 1% NaCl solution (1g NaCl +100ml distilled water, ready to use) was added, and mixed well, and filtered with filter paper.
2.1.4 Main Instrument
Other instruments and equipment are all made in China or imported in recent years and are provided by science and technology park of western hospitals of Sichuan university.
1.2 Experimental methods
1.2.1 rat Knee OA model establishment
The same age rats were 12, 3 of which served as normal controls and 9 of which were subjected to knee joint OA modeling. The modeling operation process is completed in an SPF (specific pathogen free) SD (rat) operation room of the Experimental animal center of Sichuan university, the method [4] of cutting off the medial collateral ligament and cutting off the medial meniscus is adopted, the whole operation process is strictly operated in a sterile mode, and the specific modeling method is as follows:
1) The weight of the rat is weighed before the operation, the dosage of 10 percent chloral hydrate required by anesthesia is calculated according to the dosage of 3 to 4ml/kg, and the rat is anesthetized by intraperitoneal injection.
2) After satisfactory anesthesia, the area of about 4cm above and about 2cm below and about was shaved with a shaver centered on the knee joint.
3) The prepared skin rat was placed on the operating table in a supine position, and the other limbs were fixed except for the postoperative limb.
4) The 5% povidone iodine solution is dipped by a cotton ball to disinfect the knee joint operation skin preparation area for 2 times, and a self-made sterile disposable hole towel is laid.
5) The skin was incised with a 15-gauge scalpel down the patella and medial edge of the patellar ligament in a longitudinal row for approximately 0.5-1cm long, and the subcutaneous and muscle layers were incised until the joint capsule was exposed.
6) The joint capsule is cut along the inner side of the patella and the patellar ligament by an ophthalmologic scissors, the fat pads of the anterior tibialis and the intercondylar notch are separated bluntly, and the hemostasis is pressed by a sterile cotton ball when bleeding occurs in the operation process.
7) The knee joint is straightened, the patella is pushed to the lateral side, the knee joint is bent, the anterior horn of the medial meniscus and the medial collateral ligament are fully exposed, and the sterile NS is dipped by a cotton ball to keep the joint moist in the operation process.
8) The medial collateral ligament was cut with a small sharp knife, the tibia was supinated, the anterior horn and body of the medial meniscus were fully exposed, the medial meniscus was carefully removed with an ophthalmic scissors, and the femoral condyle articular cartilage was not damaged with the sharp knife and scissors. The medial meniscus, and in particular the posterior horn of the meniscus, was examined for integrity.
9) After the medial meniscus was determined to be completely resected, povidone-iodine solution and NS were sequentially washed, the knee was straightened, the patella was repositioned, the joint capsule was continuously sutured with 5-0 absorbable thread, the skin incision was sutured intermittently with 5-0 silk thread, and the incision was again disinfected with povidone-iodine solution.
10) Injecting 1ml of penicillin (4 wu/ml) into abdominal cavity after operation to prevent infection, dividing the rat into cages according to the number after the rat is awake, putting the rat into a rat room for conventional feeding, and allowing the rat to freely move and eat water.
The rat is examined on days 1, 2 and 3 after operation, and if seepage or red swelling exists, the incision is disinfected by povidone iodine solution.
1.2.2 rat Knee OA model identification
Rats were sacrificed by means of excess anaesthesia at 4, 8 and 12 weeks after the modeling surgery, 3 were sacrificed at each time point,
3 normal rats were taken as normal controls. Knee joints were surgically exposed and the success of rat knee OA modeling was identified by gross and histological observations.
1.2.2.1 general observations
The knee joint femoral condyle was fully exposed, visually observed and the degenerative condition of the articular cartilage in the femoral condyle load bearing area was recorded with a digital camera under the same conditions. The main contents of the general observation include: the smoothness and color of the articular cartilage surface, whether the cartilage surface has cracks, defects, ulcers and degrees thereof, whether the subchondral bone is exposed, and the like.
1.2.2.2 histological section observations
(1) Specimen sampling and processing
1) After general observation is finished, quickly separating and modeling the knee joint, carefully shearing soft tissues around the joint to ensure that the femoral condyle is complete, immediately placing the knee joint in a 50ml centrifuge tube containing enough 10% paraformaldehyde, completely submerging the knee joint specimen, and fixing for 24-48 h.
2) The knee joint specimens were transferred to 50ml centrifuge tubes containing enough 20% EDTA solution, decalcified for 4 weeks, and the decalcification solution was changed once a week.
3) Taking out the knee joint specimen, trimming irregular bone tissue at the proximal end of the femoral condyle by using a clean sharp surgical blade, splitting the femoral condyle along the midline vector of the intercondylar notch, discarding the lateral condyle of the femur, keeping the medial condyle, and continuing decalcification for 2 weeks.
4) After the decalcification is finished, the mixture is washed by running water for 12 to 24 hours, acetone is dehydrated step by step, and dimethylbenzene is transparent.
5) And (5) embedding after wax dipping.
6) The sections were sectioned along the sagittal plane at intervals of 300 μm, thickness 5 μm, and stored in a refrigerator at 4 ℃ for later use.
(2) HE staining
1) The paraffin sections were rewarming at 60 ℃ for 20min and then deparaffinized by xylene (I) and xylene (II) for 10min each.
2) Sequentially washing with xylene (I) and xylene (II) for 5min, 100% ethanol for 2min, 95% → 80% → 75% ethanol for 1min, and distilled water for 1 min.
3) Soaking and staining with hematoxylin staining solution for 15min, and washing with tap water for 30 s.
4) Differentiation was carried out for 30s with 1% ethanol hydrochloride, and washing with tap water for 30 s.
5) Soaking in blue promoting solution for 10-30s, and soaking in tap water for 15min or warm water at 50 deg.C for 5 min.
6) Staining with 5% eosin staining solution for 2 min.
7) Dehydrating with 95% (I) → 95% (II) → 100% (I) → 100% (II) ethanol for 1min each.
8) Clear through xylene stone carbonic acid → xylene (I) → xylene (II) each for 1 min.
9) And (5) sealing the neutral gum.
(3) Toluidine blue staining
1) Paraffin wax slices are rewarming at 60 ℃ for 20min and then dewaxed by xylene (I) → xylene (II) for 15min each.
2) Washing with distilled water for 1min sequentially for 100% (I) → 100% (II) → 95% (I) → 95% (II) → 80% → 75% ethanol for 1min each.
3) The sections were placed in 0.5% toluidine blue aqueous solution for 10 min.
4) Flushing with running water for 2 min.
5) Acetone differentiation was stopped until osteochondral cell structures were clear.
6) Dehydrating with 70% → 80% → 90% → 95% (I) → 95% (II) → 100% (I) → 100% (II) ethanol for 1min each in sequence.
7) Passing through xylene (I) → xylene (II) in this order for 10min each, and then, clearing.
8) And (5) sealing the neutral gum.
(4) Observation of tissue sections
Each group of specimens was randomly selected from HE and toluidine blue stained histological sections of the same site for histological observation, the main observation indices included cartilage tissue structure, chondrocyte number, matrix staining and tide line integrity, and histological scoring was performed according to the modified Mankin's method (table 7).
TABLE 7 articular cartilage histology Mankin's Scale rules
1.2.3 rat Knee-Joint intracavity injection of miRNA-140agomir
after 1 week of modeling operation, performing joint cavity puncture injection under the condition that a model operation incision is basically healed, wherein the process is completed in an SPF (specific pathogen free) SD (rat) operation room of the Experimental animal center of Sichuan university, and the whole process is strictly aseptic, and the specific method comprises the following steps:
1.2.3.1 groups of Experimental animals
The part needs 42 rats in total, wherein 6 rats are used as a normal control group without any treatment, and 36 rats are randomly divided into a miRNA-140agomir group and a miRNA-140control group after OA modeling operation, and each group is 18 rats.
1.2.3.2 preparation of miRNA-140agomir injection
5nmol of miRNA-140agomir freeze-dried powder is taken out, is subjected to instantaneous centrifugation before use, and is prepared into 100 mu l of injection by using sterilized double distilled water, and the injection is prepared before each experiment. The preparation method of the miRNA-140control injection is the same.
2.2.3.3 Intra-articular injection of miRNA-140agomir
1) Before the operation, the weight of the rat is weighed, the dosage of 10 percent chloral hydrate required by anesthesia is calculated according to the dosage of 3-4ml/kg, and the rat is anesthetized by intraperitoneal injection.
2) after satisfactory anesthesia, the rat was placed on the operating table in the supine position and the remaining limbs were immobilized except for the postoperative limbs.
3) taking the middle point of the knee joint gap at the inner side of the patellar ligament as a needle insertion point, and dipping a cotton ball with 5% povidone iodine solution to disinfect the knee joint operation area.
4) The joint cavity was punctured with a micro-syringe under sterile conditions and 100. mu.l of miRNA-140agomir or miRNA-140control injection was injected.
5) Pressing with sterile cotton ball for 2-5min to prevent leakage of injection.
After the rats are awake, the rats are placed in cages according to the numbers and are put back into the rat room for conventional feeding, and the rats are allowed to move freely and take food and water.
1.2.4 detection of Effect of miRNA-140 injected into articular Cavity on articular cartilage degeneration
As described above, 6 rats were sacrificed at 4, 8 and 12 weeks after the model building in the miRNA-140agomir group and the miRNA-140control group, and 6 normal rats were used as controls. The knee joint is exposed by operation, and the action effect and the molecular mechanism of the miRNA-140agomir injected into the joint cavity on the rat knee joint cartilage degeneration are detected by gross observation, pathological tissue section staining and immunohistochemical staining methods.
1.2.4.1 general observations
The knee joint femoral condyle was fully exposed, visually observed and the degenerative condition of the articular cartilage in the femoral condyle load bearing area was recorded with a digital camera under the same conditions. The main contents of the general observation include: the smoothness and color of the articular cartilage surface, whether the cartilage surface has cracks, defects or ulcers and the degree thereof, whether subchondral bone is exposed, and the like.
1.2.4.2 histological section observations
(1) HE staining
The specific method is the same as the previous method.
(2) Toluidine blue staining
The specific method is the same as the previous method.
(3) mankin's histological score
The specific method is the same as the previous method.
1.2.4.3 immunohistochemical observations
And taking a tissue section for immunohistochemical staining, observing the expression conditions of Collagen II, MMP13 and ADAMTS-5 proteins in the cartilage tissue, and carrying out semi-quantitative analysis on the expression of the corresponding proteins by calculating the average optical density value of a positive area.
(1) Immunohistochemical method
1) and (3) placing the paraffin section in an oven at 60 ℃ for baking for 20-30min for rewarming.
2) Sequentially passing through xylene (I) → xylene (II) for 20min, sequentially passing through 100% → 95% → 80% → 70% ethanol for 5min, washing with running water for 5min, and washing with distilled water for 1 time.
3) PBS wash 3 times for 5min each.
4) An appropriate amount of 3% H2O2 was added dropwise to each section, and incubation was performed at room temperature for 10min to inactivate endogenous peroxidase.
5) PBS wash 3 times for 5min each.
6) Add 30. mu.l of primary antibody at the appropriate dilution, cover the tissue well, and place in a wet box overnight in a refrigerator at 4 ℃.
7) Rewarming in an oven at 37 deg.C for 20min, washing with PBS for 3 times, 5min each time.
8) PBS was removed, 30. mu.l of secondary antibody working solution was added dropwise to each section, and incubation was carried out at room temperature for 30 min.
9) PBS wash 3 times for 5min each.
10) PBS is removed, 30 mul of freshly prepared DAB is dripped into each section, the section is dyed for 5-10min at room temperature, the dyeing degree is observed under a microscope, the dyeing is stopped timely, and the section is washed for 30min by tap water.
11) Counterstaining with hematoxylin staining solution for 5min, differentiating with 0.5% hydrochloric acid ethanol for 10s, washing with tap water for 15min, and turning blue.
12) Sequentially passing through 95% ethanol (I) → 95% ethanol (II) for 5min respectively, toning and dehydrating, sequentially passing through 100% ethanol (I) → 100% ethanol (II) → xylene (I) → xylene (II) for 5min respectively, washing with tap water for 5min, and making the transparent product.
13) And (5) sealing the neutral gum.
(2) Immunohistochemical results analysis
Each immunohistochemical staining section was photographed and stored under the same condition, and Image-Pro Plus (IPP)6.0 software was used to detect the total area (area) and total Integrated optical density (iod) (sum)) of the cartilage layer positive area and the average optical density (MOD) of the iod (sum)/area (sum)) of each Image under the same setting, and the protein expression was semi-quantitatively analyzed. And selecting 3 positive areas for each sample, carrying out detection calculation, then averaging, and finally, applying statistical software to carry out statistical analysis on the difference conditions among the groups.
1.3 statistical analysis
Data were collected using Excel and statistically analyzed using SPSS 22.0 software. Comparison between groups Using the Mann-Whitney U test, p <0.05 was considered statistically significant.
2 results
2.1 rat Knee OA model identification
2.1.1 general conditions after modeling rat Knee OA
The mean time of knee OA modeling operation of rats is 10.60 +/-1.80 min, all rats are awake within 2h after operation, and operation incisions are healed within 1 week after operation, so that no joint infection and no dead rats exist.
2.1.2 general observations
3 rats were sacrificed at weeks 4, 8 and 12 after the modeling operation, and 3 normal rats were taken as normal controls. After exposing the femoral condyle, the degree of articular cartilage degeneration is observed by naked eyes and recorded by taking a picture by a digital camera, and typical articular cartilage images are obtained at various time points.
Normal rats: the femoral condyle cartilage has smooth surface and bright color.
4 weeks after surgery: the cartilage of the medial condyle of the femur has rough surface and light color, no definite damage and ulcer formation, and no obvious osteophyte formation.
8 weeks after surgery: articular cartilage in the medial femoral condyle load-bearing area is obviously abraded, ulcer, cartilage defect, subchondral bone exposure and the like appear on part of joint surfaces of rats, and osteophyte formation is seen around the joint surfaces.
12 weeks after surgery: the medial femoral condyle articular cartilage was severely worn, subchondral bone was exposed, and significant osteophyte formation was seen around the subchondral cartilage.
2.1.3 histochemical results
Pathological sections of the medial femoral condyle were taken and stained with HE and toluidine blue, and cartilage degeneration was evaluated using Mankin's histological scoring, with the following results.
(1) HE staining
HE stain images were typical for each time point.
Knee cartilage of normal rat: after HE staining, the cartilage matrix is uniformly stained and pink, and the chondrocytes are regularly distributed and blue. Articular cartilage has a well-defined tissue structure and can be divided into superficial, transitional, radial and calcified layers. The surface layer cartilage cells have small volume and are arranged in a flat shape; cartilage cells of the transitional layer and the radiation layer gradually become larger and round and are arranged sparsely; the calcified layer cartilage cells have the largest volume and are arranged close to the tide line in a columnar-like shape.
4 weeks after surgery: the covering of fibrous tissues on the surface of cartilage is obvious, cells on the surface layer and the middle layer shrink, cartilage cells on partial articular surface fall off, the cartilage layer becomes thin, the cartilage cells are increased in a diffused manner on the whole, and a large number of clustered cell clusters are accompanied, so that the tide lines are fuzzy, and the cartilage cells are partially broken.
8 weeks after surgery: the articular cartilage is destroyed to the interface of the calcified layer and the subchondral bone, the number of chondrocytes is reduced, and necrotic disintegrating cells, disorganized and broken structures of the hygrometric line can be seen.
12 weeks after surgery: the destruction of the articular cartilage is further aggravated, the cartilage is destroyed to the subchondral bone layer, the subchondral bone is exposed, the damaged area of the articular surface is covered by fibers, few chondrocytes remain, and the tide line disappears.
(2) Toluidine blue staining
A typical toluidine blue stained image at each time point.
Knee cartilage of normal rat: after toluidine blue staining, the cartilage matrix is stained uniformly and in dark blue, and the chondrocytes are regularly distributed. The articular cartilage tissue structure is clear.
4 weeks after surgery: the covering of fibrous tissues on the surface of cartilage is obvious, cells on the surface layer and the middle layer shrink, partial cells fall off, the cartilage layer becomes thin, the number of chondrocytes is increased, clustered cell clusters are accompanied, the tide lines are blurred, partial cells are broken, and the staining of cartilage matrixes is slightly to moderately reduced.
8 weeks after surgery: the abrasion of articular cartilage is aggravated, the cartilage surface is damaged, part of the articular cartilage can reach the radiation layer, the number of chondrocytes is reduced, the hygrometric structure is disordered and broken, and the coloring degree of the cartilage matrix is reduced to be severe.
12 weeks after surgery: the articular cartilage destruction is further aggravated, few chondrocytes remain, cartilage is destroyed to a calcified layer or even a subchondral bone junction, large area of subchondral bone is exposed, the tide line disappears, and the residual matrix is severely colored and is reduced or even not colored.
(3) Mankin's histological score
The rats knee cartilage Mankin's histological scores at different time points after the modeling are shown in table 8. Normal rat articular cartilage Mankin's score was 0, 6.50 ± 1.52, 10.17 ± 0.75 and 12.83 ± 0.75 weeks post-modeling, respectively, at 4, 8 and 12 weeks post-modeling.
TABLE 8 comparison of the Mankin's histological scores of the cartilage of the knee joint of two groups of rats at different time points after modeling
2.2 Effect of miRNA-140 articular cavity injection on rat knee cartilage degeneration
Rat knee OA modeling the surgical incision was substantially healed at 1 week post-surgery (as before), at which time after anesthetizing the rat, the rat was observed for general conditions by injecting 50nmol/100 μ l miRNA-140agomir or miRNA-140control via joint cavity puncture. Two groups of 6 dead rats each at weeks 4, 8 and 12 after the model surgery were used as controls, 6 normal rats. Rat articular cartilage degeneration after miRNA-140agomir and miRNA-140control articular cavity injection is observed and compared through gross observation, tissue section staining and immunohistochemical staining.
2.2.1 general conditions in rats after articular cavity injection of miRNA-140
All rats were awake within 2h after the joint cavity puncture. No complications such as joint infection or death of rats with miRNA-140agomir or miRNA-140control injected into joint cavities occur until the experiment is finished. Therefore, the miRNA-140 can be safely and practically injected into the joint cavity.
2.2.2 general observations
Two sets of representative articular cartilage images at various time points. The cartilage surface of the femoral condyle of a normal rat is smooth and bright in color.
After 4 weeks of miRNA-140control group surgery, the surface of medial femoral condyle cartilage becomes rough and becomes dark in color; the articular cartilage in the medial femoral condyle load bearing area is obviously abraded at 8 weeks after operation, and part of the articular cartilage is ulcerated, cartilage is damaged and osteophyte is formed; after 12 weeks of operation, the cartilage of the medial femoral condyle joint is seriously abraded, subchondral bone is exposed in a large area, and obvious osteophyte formation is visible around the subchondral bone.
The surface of the medial femoral condyle cartilage is still smooth 4 weeks after the miRNA-140agomir group operation, the color is dark, the joint surface is not damaged and ulcer and no obvious osteophyte is formed; the articular cartilage surface of the medial femoral condyle load bearing area is slightly rough after 8 weeks of operation, and small ulcer and osteophyte are occasionally formed; at 12 weeks postoperatively, the articular cartilage of the medial femoral condyle wears away, has a rough surface, and is partially affected by ulceration and osteophyte formation. Therefore, it was generally observed that the articular cavity injection of miRNA-140agomir could significantly delay the progression of articular cartilage degeneration in rats.
2.2.3 histological results
(1) HE staining
two sets of representative HE stain images at each time point. After HE staining is carried out on knee joint cartilage of a normal rat, cartilage matrix staining is uniform, chondrocyte distribution is regular, and the articular cartilage tissue structure is clear.
After 4 weeks of miRNA-140control operation, fibrous tissue covering is visible on the surface of the cartilage, the cartilage layer becomes thin, partial cells shrink, cartilage cells on the joint surface fall off, the cartilage cells increase on the whole, and are accompanied by clustered cell clusters, the tide lines are fuzzy, and partial cartilage cells break; articular cartilage destruction can be seen at 8 weeks after operation, and part of the articular cartilage destruction reaches the calcified layer or the subchondral bone junction, so that the number of cells in the cartilage layer is obviously reduced, necrotic cells can be seen, and the hygrometric structure is disordered and broken; the articular cartilage destruction is further aggravated after 12 weeks of operation, the cartilage layer is worn completely, few chondrocytes remain, subchondral bone is exposed, the visible fiber covering of the damaged area of part of the articular surface is realized, and the tide line disappears.
After 4 weeks of the miRNA-140agomir group operation, the cartilage structure is basically normal, the chondrocytes are not obviously changed or slightly increased, no obvious cell clustering phenomenon is found, and the tide line is complete; the cartilage layer becomes slightly thinner and rough at 8 weeks after operation, the number of chondrocytes is normal or slightly reduced, and part of cartilage layer is visible with clustered cell clusters and part of tide lines are disordered; the articular cartilage is damaged and aggravated at 12 weeks after the operation, part of the surface layer falls off, the number of chondrocytes is obviously reduced, and the structure of the damp line is disordered and broken.
(2) Toluidine blue staining
Two groups stained images with typical toluidine blue at each time point. After the cartilage of the knee joint of a normal rat is stained by toluidine blue, the cartilage matrix is uniformly stained, the chondrocyte distribution is regular, and the articular cartilage tissue structure is clear.
After 4 weeks of miRNA-140control group operation, the increase of the number of the chondrocytes is observed with cluster cell masses, and the staining of cartilage matrixes is slightly to moderately reduced; the articular cartilage layer is further thinned at 8 weeks after the operation, the cartilage destruction is further deepened, the number of cartilage cells is obviously reduced, the staining severity of the cartilage matrix is reduced, and the partial severity is reduced; the cartilage layer is almost completely worn at 12 weeks after operation, few chondrocytes remain, large area of subchondral bone is exposed, and the staining severity of the residual matrix is reduced or even no staining is caused.
The number of chondrocytes is not obviously reduced in the miRNA-140agomir group at 4 weeks, and the cartilage matrix is slightly reduced after being normally stained; the articular cartilage layer becomes thin at 8 weeks after the operation, the number of chondrocytes is reduced, and the coloring of cartilage matrix is reduced from light to moderate; the cartilage wear is increased at 12 weeks after the operation, the cartilage layer becomes thinner, the surface is rough, the number of cells is reduced, and the medium-to-severe degree of matrix staining is reduced.
(3) Mankin's histological score
The histological scores of Mankin's at different time points in the two groups are shown in Table 9, the Mankin's score of the miRNA-140agomir group is obviously lower than that of the control group at each time point, and the difference is statistically significant (p < 0.05). As can be seen, the histological observation further proves that the joint cavity injection of the miRNA-140agomir can obviously delay the degeneration process of the rat articular cartilage.
TABLE 9 comparison of articular cartilage Mankin's histological scores of two groups of rats at different time points
2.2.4 immunohistochemical results
6 dead rats at 4, 8 and 12 weeks after OA modeling surgery are taken, the medial condyle of the femur is subjected to tissue section and then subjected to immunohistochemical detection, the expression conditions of Collagen II, MMP13 and ADAMTS-5 proteins in cartilage tissues are observed under a light mirror, the positive expression is brownish yellow particles, the average optical density value (MOD) of a positive region is calculated through IPP6.0 software to realize semi-quantitative analysis of protein expression, and the protein expressions of the miRNA-140agomir group, the miRNA-140control group Collagen II, MMP13 and ADAMTS-5 are compared.
(1) Regulation effect of miRNA-140agomir injected into joint cavity on expression of Collagen II in cartilage tissue
The expression condition of Collagen II protein in cartilage tissue is observed under a light mirror, and a large amount of brown-yellow positive expression can be seen in normal rat cartilage tissue and is mainly positioned on a surface layer, a transitional layer and a radiation layer.
The miRNA-140control group obviously weakens the positive expression at 4 weeks after operation, and the transition layer and the radiation layer are more obviously weakened; the positive expression is further weakened at 8 weeks after the operation, and a small amount of positive expression can be seen only on the damaged cartilage surface layer and the transitional layer; at 12 weeks after surgery, the cartilage layer had disappeared and only a very small number of chondrocytes remained with a weak positive expression.
The miRNA-140agomir group still can show stronger positive expression in 4 weeks after operation; the positive expression is weakened at 8 weeks after operation, and the expression of the radiation layer is mainly weakened; positive expression was further reduced at 12 weeks after surgery, but some positive expression was still seen in the irregular surface layer of cartilage.
MOD quantitative analysis results suggest that MOD values at each time point after the miRNA-140agomir group operation are significantly higher than those of the miRNA-140control group, and the differences have statistical significance (Table 10). Thus, the expression of Collagen II in the cartilage tissue is gradually reduced along with the progressive degeneration degree of the articular cartilage; and the loss of Collagen II protein in cartilage tissues can be remarkably slowed down by injecting miRNA-140agomir into the joint cavity in the process of joint cartilage degeneration.
TABLE 10 comparison of Collagen II expression in cartilage tissue of two groups of rats at different time points (MOD,. times.10-3)
(2) Regulating and controlling effect of miRNA-140agomir injected into articular cavity on MMP13 expression in cartilage tissue
MMP13 protein expression in cartilage tissue is observed under a light mirror, and a small amount of brown yellow positive expression can be seen in normal rat cartilage tissue and is scattered in a migration layer and a radiation layer.
The miRNA-140control group obviously enhances the positive expression at 4 weeks after operation, and the deep layer is obviously enhanced; the positive expression is further enhanced at 8 weeks after operation, and strong positive expression can also be seen in the damaged articular surface; at 12 weeks after surgery, the cartilage layer was severely abraded and strong positive expression was seen in the remaining cartilage tissue.
The miRNA-140agomir group can show more obvious positive expression at 4 weeks after operation, but is lower than the control group; the positive expression is enhanced at 8 weeks after operation, and the cartilage deep layer is enhanced more obviously; the positive expression of the cartilage superficial layer is enhanced at 12 weeks after the operation.
the MOD quantitative analysis result indicates that the MOD value of each time point after the miRNA-140agomir group is obviously lower than that of the miRNA-140control group, and the difference has statistical significance (Table 11). Thus, MMP13 is expressed at a low level in normal cartilage tissue, and the MMP13 expression in the cartilage tissue is gradually increased along with the progressive degradation degree of the articular cartilage; and the miRNA-140agomir injected into the joint cavity in the process of articular cartilage degeneration can obviously inhibit the MMP13 protein expression in the cartilage tissue.
TABLE 11 comparison of MMP13 expression in two groups of rat cartilage tissues at different time points (MOD,. times.10-3)
(3) Regulating and controlling effect of miRNA-140agomir injected into articular cavity on ADAMTS-5 expression in cartilage tissue
A small number of brown-yellow particles are expressed in normal rat cartilage tissue and are scattered in the whole cartilage tissue layer.
The miRNA-140control group obviously enhances the positive expression at 4 weeks after operation, and has an obvious surface layer; the positive expression is further enhanced at 8 weeks after operation, and the superficial expression is stronger than the deep expression; at 12 weeks after surgery, the cartilage layer was significantly thinned and strong positive expression was seen in the remaining cartilage tissue.
The miRNA-140agomir group has enhanced positive expression at 4-8 weeks after operation and is mainly concentrated in the deep layer; the expression of the whole layer of cartilage is obviously enhanced at 12 weeks after the operation.
MOD quantitative analysis results suggest that the MOD value at each time point after the miRNA-140agomir group is significantly lower than that of the miRNA-140control group, and the difference is statistically significant (p <0.05) (Table 12). Thus, ADAMTS-5 is expressed at a low level in normal cartilage tissue, and the ADAMTS-5 expression in the cartilage tissue gradually increases as the degeneration degree of the articular cartilage increases; and the expression of ADAMTS-5 protein in cartilage tissue can be obviously inhibited by injecting miRNA-140agomir into the joint cavity in the process of articular cartilage degeneration.
TABLE 12 comparison of ADAMTS-5 expression in two groups of rat cartilage tissues at different time points (MOD,. times.10-3)
Articular cartilage degeneration is one of the main features of OA, and mainly involves degradation of ECM such as Collagen type ii (Collagen ii) and proteoglycans (agrrechans). MMP-13 and ADAMTS-5 are the most important hydrolases in matrix degrading enzymes, L finds that miRNA-140 is a negative regulatory factor of MMP13, and miRNA-140 can inhibit MMP13 expression in the process of using proinflammatory factor IL-1 beta to induce chondrocyte OA-like change; they further use luciferase reporter gene analysis to find that miRNA-140 can directly combine with UTR site at MMP 133' end. M and the like find that the intervention of proinflammatory factor IL-1 beta can reduce the expression of miRNA-140 of chondrocytes and increase the expression of ADAMTS-5 in vitro experiments; when miRNA-140 expression is increased by gene transfection, the induction of IL-1 beta is obviously weakened. Further, it was found that ADAMTS-5 expression was increased in miRNA-140-/-mice and decreased in miRNA-140 transgenic mice, and in vitro luciferase reporter gene analysis also found that the UTR at the' end of ADAMTS-53 contained miRNA-140 binding sites. Therefore, ADAMTS-5 expression inhibition is also one of the mechanisms by which miRNA-140 prevents articular cartilage degeneration. In the invention, the exogenous miRNA-140 is injected through articular cavity puncture, and the result shows that the loss of Collagen II in articular cartilage is obviously slower than that of a control group in the induction process of a rat OA model, and the expression of MMP13 and ADAMTS-5 is obviously inhibited, which is consistent with the results of cell experiments in the first part and previous transgenic/gene knockout animals. Thus, it can also be concluded that: in the experiment, the effect of the miRNA-140agomir injected into the articular cavity to delay the degeneration of the articular cartilage is realized at least in part by inhibiting the expression of MMP13 and ADAMTS-5 in the articular cartilage.
The invention successfully establishes a rat OA model, and the joint cavity injection of the miRNA-140agomir is safe and feasible.
The joint cavity injection of miRNA-140agomir can obviously slow down the degeneration process of rat articular cartilage.
The miRNA-140agomir injected into the articular cavity can obviously slow down the loss of Collagen II protein in articular cartilage tissues, obviously inhibit the expression of MMP13 and ADAMTS-5 protein and obviously delay the degeneration of rat articular cartilage.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (1)
- Use of miRNA-140in the manufacture of a medicament for slowing the progression of articular cartilage degeneration;The medicament is used for slowing down the loss of Collagen II protein in articular cartilage tissues and inhibiting the expression of MMP13 and ADAMTS-5 protein.
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