阅读说明：本技术 一种LncRNA LOC102555148的应用 (Application of LncRNA LOC102555148 ) 是由 张飞 彭吾训 王强 王涛 徐凤阳 张健 董文涛 于 2020-12-29 设计创作，主要内容包括：本发明提供了一种LncRNA LOC102555148的应用,LncRNA LOC102555148用于制备抑制BMSCs缺氧性凋亡,进而提高BMSCs的移植疗效的药物。本发明中LncRNA LOC102555148在BMSCs缺氧性凋亡中起保护作用,LncRNA LOC102555148通过抑制BMSCs缺氧性凋亡,促进BMSCs在缺氧环境下的存活,从而提高BMSCs的移植疗效。因此,LncRNA LOC102555148可用于制备抑制BMSCs缺氧性凋亡的药物,具有广阔的临床应用前景。(The invention provides an application of LncRNA LOC102555148, and the LncRNA LOC102555148 is used for preparing a medicament for inhibiting hypoxic apoptosis of BMSCs and further improving the transplantation curative effect of the BMSCs. In the invention, LncRNA LOC102555148 plays a protective role in BMSCs hypoxic apoptosis, and LncRNA LOC102555148 promotes the BMSCs to survive in a hypoxic environment by inhibiting BMSCs hypoxic apoptosis, thereby improving the transplantation curative effect of the BMSCs. Therefore, LncRNA LOC102555148 can be used for preparing medicines for inhibiting BMSCs hypoxic apoptosis and has wide clinical application prospect.)

1. The application of LncRNA LOC102555148 is characterized in that the LncRNA LOC102555148 is used for preparing a medicine for inhibiting hypoxic apoptosis of BMSCs and further improving the transplantation curative effect of the BMSCs.

2. The use of LncRNA LOC102555148, as claimed in claim 1, wherein the medicament comprises a plasmid comprising LncRNA LOC102555148 sequence or a lentiviral expression vector comprising LncRNA LOC102555148 sequence.

3. The use of LncRNA LOC102555148 in claim 1 or 2, wherein the LncRNA LOC102555148 has a nucleotide sequence of seq id No.1 of the sequence listing.

Technical Field

The invention belongs to the technical field of LncRNA, and particularly relates to an application of LncRNA LOC 102555148.

Background

Bone marrow mesenchymal stem cells (BMSCs) have a strong regenerative capacity and have been used for transplantation therapy of various ischemic and hypoxic diseases. However, transplanted BMSCs have high apoptosis in hypoxic microenvironment of focal region, so that the transplantation curative effect is limited, and how to inhibit hypoxic apoptosis of BMSCs is the key for improving the transplantation curative effect. At present, no medicine for preventing the anoxic apoptosis of BMSCs exists.

Long non-coding RNA (LncRNA) is a non-coding RNA molecule with the transcript length of 200nt-100kb, and can regulate the expression of apoptosis-related genes in a sequence-specific manner so as to intervene in various life activities such as apoptosis and the like. The application of LncRNA in inhibiting BMSCs hypoxic apoptosis is still in the development stage, and needs continuous exploration and research.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an application of LncRNA LOC102555148 aiming at the defects of the prior art, the LncRNA LOC102555148 plays a role in BMSCs hypoxic apoptosis, and the LncRNA LOC102555148 promotes the BMSCs survival in a hypoxic environment by inhibiting the BMSCs hypoxic apoptosis, so that the transplantation curative effect of the BMSCs is improved. Therefore, LncRNA LOC102555148 can be used for preparing medicines for inhibiting BMSCs hypoxic apoptosis and has wide clinical application prospect.

In order to solve the technical problems, the invention adopts the technical scheme that: an application of LncRNA LOC102555148, wherein the LncRNA LOC102555148 is used for preparing a medicament for inhibiting hypoxic apoptosis of BMSCs and further improving transplantation curative effect of the BMSCs.

Preferably, the medicament comprises a plasmid comprising LncRNA LOC102555148 sequence or a lentiviral expression vector comprising LncRNA LOC102555148 sequence.

Preferably, the LncRNA LOC102555148 has a nucleotide sequence of seq.id No.1 of the sequence listing.

Compared with the prior art, the invention has the following advantages:

the LncRNA LOC102555148 plays a role in protecting BMSCs from hypoxic apoptosis, and the LncRNA LOC102555148 promotes the BMSCs to survive in the hypoxic environment by inhibiting the BMSCs from hypoxic apoptosis, so that the transplantation curative effect of the BMSCs is improved. Therefore, LncRNA LOC102555148 can be used for preparing medicines for inhibiting BMSCs hypoxic apoptosis and has wide clinical application prospect.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Drawings

FIG. 1 is a graph showing the detection of hypoxic apoptosis of BMSCs by Annexin V-FITC/PI double staining after the LncRNA LOC102555148 is knocked out in example 1 of the present invention.

FIG. 2 is a graph showing the detection of apoptosis of BMSCs by TUNEL staining after the deletion of LncRNA LOC102555148 in example 1 of the present invention.

FIG. 3 is a graph showing the detection of hypoxic apoptosis of BMSCs by Annexin V-FITC/PI double staining after over-expression of LncRNA LOC102555148 in example 1 of the present invention.

FIG. 4 is a graph showing the detection of hypoxic apoptosis of BMSCs by TUNEL staining after over-expression of LncRNA LOC102555148 in example 1 of the present invention.

Fig. 5 is a graph of GFP immunofluorescence detecting apoptosis of transplanted BMSCs in a femoral head necrosis zone hypoxic environment 48 hours after transplantation of BMSCs in example 2 of the present invention.

Fig. 6 is a graph of apoptosis of transplanted BMSCs in hypoxic environment in femoral head necrosis region, measured by TUNEL staining 48 hours after transplantation of BMSCs in example 2 of the present invention.

Fig. 7 is a view of the femoral head necrosis area repaired in a gross specimen observation 12 weeks after the BMSCs transplantation in example 2 of the present invention.

Fig. 8 is a graph showing the area of new bone formation in the femoral head necrosis area detected by HE staining 12 weeks after the BMSCs transplantation in example 2 of the present invention.

Fig. 9 is a graph of new bone formation in femoral head necrosis zones detected by Masson staining 12 weeks after BMSCs implantation in accordance with example 2 of the present invention.

Detailed Description

Example 1

This example is the use of LncRNA LOC102555148 for in vitro experiments, said LncRNA LOC102555148 is used for the preparation of a medicament for inhibiting hypoxic apoptosis of BMSCs comprising a plasmid comprising the LncRNA LOC102555148 sequence or a lentiviral expression vector comprising the LncRNA LOC102555148 sequence;

the LncRNA LOC102555148 has a nucleotide sequence of a sequence table SEQ.ID.No.1;

the LncRNA LOC102555148 is long-chain non-coding RNA LOC102555148, and the BMSCs are bone marrow mesenchymal stem cells.

In vitro experiments:

test A:

separating and culturing primary BMSCs, knocking out LncRNA LOC102555148 of the BMSCs by using CRISPR/Cas9 technology, and then carrying out hypoxia (0% oxygen, 95% nitrogen and 5% carbon dioxide) treatment on the BMSCs for 48 hours, wherein the experiment is divided into 3 groups: the apoptosis of BMSCs in each group is detected by a TUNEL staining method and an Annexin V-FITC/PI double staining method, and experiments show that the apoptosis rate of BMSCs knocked out by the LncRNA LOC102555148 is remarkably increased (the number of TUNEL and Annexin V-FITC/PI positive cells is increased) compared with that of an LncRNA LOC102555148 knocked out by the non-LncRNA LOC102555148 (hypoxia) group.

As shown in FIG. 1, Annexin V-FITC/PI double staining method detects apoptosis of BMSCs in each group, wherein a is an unbaked LncRNA LOC102555148 (normal oxygen concentration), b is an unbaked LncRNA LOC102555148 (hypoxia), and c is a knocked LncRNA LOC102555148 (hypoxia). As shown in FIG. 2, TUNEL staining method detects apoptosis of BMSCs in each group, wherein a is an undeleted LncRNA LOC102555148 (normal oxygen concentration), b is an undeleted LncRNA LOC102555148 (hypoxia), and c is a knocked-out LncRNA LOC102555148 (hypoxia). As can be seen from the figure, the apoptosis rate of BMSCs cultured in normal oxygen concentration is about (6.11 +/-2.15)%, the apoptosis rate of BMSCs after hypoxia is about (50.55 +/-2.86)%, when LncRNA LOC102555148 of BMSCs is knocked out, the apoptosis rate is further increased to (65.48 +/-3.01)%, the difference is obvious, and the statistical significance is achieved (P < 0.001). This experiment demonstrates that knockout of LncRNA LOC102555148 increases hypoxic apoptosis in BMSCs.

Test B:

primary BMSCs were isolated and cultured, transfected with LncRNA LOC102555148 overexpression lentivirus, and then treated with hypoxia (0% oxygen, 95% nitrogen, and 5% carbon dioxide) for 48 hours, and the experiments were divided into 3 groups: the apoptosis of BMSCs in each group is detected by a TUNEL staining method and an Annexin V-FITC/PI double staining method, and experiments show that the apoptosis rate of BMSCs over-expressing LncRNA LOC102555148 is remarkably reduced after suffering from hypoxia (the number of TUNEL and Annexin V-FITC/PI positive cells is reduced) compared with that of a group not over-expressing LncRNA LOC102555148 (hypoxia).

As shown in FIG. 3, Annexin V-FITC/PI double staining method detects apoptosis of BMSCs in each group, wherein a is non-overexpression LncRNA LOC102555148 (normal oxygen concentration), b is non-overexpression LncRNA LOC102555148 (hypoxia), and c is overexpression LncRNA LOC102555148 (hypoxia). As shown in FIG. 4, TUNEL staining method was used to detect apoptosis of BMSCs in each group, wherein a is under-expressed LncRNA LOC102555148 (normal oxygen concentration), b is under-expressed LncRNA LOC102555148 (hypoxia), and c is under-expressed LncRNA LOC102555148 (hypoxia). As can be seen from the figure, the apoptosis rate of BMSCs cultured in normal oxygen concentration is about (7.08 +/-1.96)%, the apoptosis rate of BMSCs after hypoxia is about (50.57 +/-2.13)%, when BMSCs overexpress LncRNA LOC102555148, the hypoxia treatment is carried out, the apoptosis rate is reduced to (17.85 +/-2.66)%, the difference is obvious, and the statistical significance is achieved (P < 0.001). This experiment demonstrates that LncRNA LOC102555148 inhibits hypoxic apoptosis of BMSCs.

Therefore, LncRNA LOC102555148 is used for preparing a medicament for inhibiting apoptosis of BMSCs comprising a plasmid comprising LncRNA LOC102555148 sequence or a lentiviral expression vector comprising LncRNA LOC102555148 sequence, for inhibiting apoptosis of BMSCs.

Example 2

The embodiment is an application of Lnc LOC102555148, and in an in vivo experiment, the Lnc LOC102555148 is used for preparing a medicament for inhibiting hypoxic apoptosis of BMSCs and further promoting survival of transplanted BMSCs in a hypoxic environment of a femoral head necrosis area, so that the curative effect of transplanting the BMSCs on the femoral head necrosis is remarkably improved, wherein the medicament comprises a plasmid containing an LncRNA LOC102555148 sequence or a lentivirus expression vector containing the LncRNA LOC102555148 sequence;

the LncRNA LOC102555148 has a nucleotide sequence of a sequence table SEQ.ID.No.1;

test A:

primary BMSCs were first isolated and cultured, LncRNA LOC102555148 of BMSCs was knocked out in vitro using CRISPR/Cas9 technology, or BMSCs were transfected with LncRNA LOC102555148 overexpression lentiviruses to overexpress LncRNA LOC102555148 and all BMSCs were labeled with Green Fluorescent Protein (GFP). Then, an SD rat femoral head necrosis model is established, and the oxygen concentration of a femoral head necrosis area is detected to be about 0 percent, so that the femoral head necrosis area can be used as an in-vivo anoxic environment. Finally, BMSCs with LncRNA LOC102555148 knocked out or over-expressed are implanted into the anoxic environment of the femoral head necrosis area, and the experiment is divided into 3 groups: BMSCs implanted with LncRNA LOC102555148 knocked out, BMSCs implanted with LncRNA LOC102555148 overexpressed. After 48 hours of operation, femoral head tissues are taken out, GFP immunofluorescence staining and TUNEL staining are adopted to detect the apoptosis condition of each group of transplanted BMSCs in femoral head necrosis areas, and experiments show that BMSCs implanted with BMSCs and implanted with knock-out LncRNA LOC102555148 are subjected to massive apoptosis in the anoxic environment of femoral head necrosis areas, while BMSCs over-expressing LncRNA LOC102555148 are remarkably reduced in the apoptosis rate in the anoxic environment of femoral head necrosis (the number of TUNEL positive cells is reduced, and the number of GFP positives is increased). After 12 weeks of operation, femoral head tissues are taken out, the femoral head tissues are longitudinally cut open, and the repair condition of femoral head necrosis areas of each group is evaluated through general specimen observation, HE staining and Masson staining, and experiments show that the necrosis areas of BMSCs which are not implanted with BMSCs, implanted with BMSCs and implanted with knock-out LncRNA LOC102555148 have no obvious repair, no new bone trabecula and mature bone tissues are seen in the necrosis areas, the necrosis areas of BMSCs which are implanted with LncRNA LOC102555148 are completely repaired, the bone trabecula is continuous and is completely reconstructed, and the bone tissues tend to be mature.

As shown in FIG. 5, at 48 hours after the operation, GFP immunofluorescence staining was used to detect the apoptosis of transplanted BMSCs in each group in the femoral head necrosis area, wherein a is BMSCs implanted, b is BMSCs over-expressing LncRNA LOC102555148, and c is BMSCs knocked out LncRNA LOC 102555148. As shown in FIG. 6, 48 hours after the operation, apoptosis of BMSCs in each group was detected by TUNEL staining, wherein a is BMSCs implanted, b is BMSCs over-expressing LncRNA LOC102555148, and c is BMSCs knocked-out LncRNA LOC 102555148. As can be seen from FIGS. 5 to 6, when BMSCs were implanted into the hypoxic environment of the necrotic area in femur, a large number of cells were apoptotic, whereas when BMSCs overexpressing LncRNA LOC102555148 were implanted into the hypoxic environment of the necrotic area in femur, the apoptosis rate was decreased, and when BMSCs knock-out LncRNA LOC102555148 were implanted into the hypoxic environment of the necrotic area in femur, the apoptosis rate was increased (the number of TUNEL-positive cells was increased, the number of GFP-positive cells was decreased)

As shown in fig. 7, femoral head tissues are taken out 12 weeks after surgery, the femoral head tissues are longitudinally cut open, and the repair condition of femoral head necrosis areas of each group is observed through a general specimen, wherein a is implanted with BMSCs, b is implanted with BMSCs over-expressing LncRNA LOC102555148, and c is implanted with BMSCs knocking out LncRNA LOC 102555148. As shown in fig. 8, femoral head tissues are taken out 12 weeks after surgery, and the repair of femoral head necrosis areas in each group is evaluated by HE staining, wherein a is BMSCs implanted, b is BMSCs over-expressing LncRNA LOC102555148, and c is BMSCs knocking LncRNA LOC102555148 out. As shown in fig. 9, femoral head tissues are taken out 12 weeks after surgery, and Masson staining is adopted to evaluate the repair of femoral head necrosis areas in each group, wherein in fig. 9, a is BMSCs implanted, b is BMSCs over-expressing LncRNA LOC102555148, and c is BMSCs knocking LncRNA LOC102555148 out. As can be seen from FIGS. 7-9, in the BMSCs group implanted with BMSCs and implanted with LncRNA LOC102555148, no obvious repair is made in the necrotic area, no new bone trabecula and mature bone tissue are found in the necrotic area, while in the BMSCs group implanted with LncRNA LOC102555148 which is over-expressed, the necrotic area is completely repaired, the bone trabecula is continuous and complete in reconstruction, and the bone tissue is mature.

Therefore, LncRNA LOC102555148 is used for preparing a medicament for promoting the survival of transplanted BMSCs in an anoxic environment of a femoral head necrosis area so as to remarkably improve the transplanting curative effect of the BMSCs on femoral head necrosis, and the medicament contains a plasmid containing LncRNA LOC102555148 sequence or a lentivirus expression vector containing LncRNA LOC102555148 sequence and is used for repairing and reconstructing bone tissues.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.

Sequence listing

<110> affiliated hospital of Guizhou medical university

<120> application of LncRNA LOC102555148

<130> 2020.11.15

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 289

<212> DNA

<213> Artificial Synthesis (Artificial Synthesis)

<400> 1

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