WO2021093401A1 - LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用 - Google Patents
LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用 Download PDFInfo
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- WO2021093401A1 WO2021093401A1 PCT/CN2020/110736 CN2020110736W WO2021093401A1 WO 2021093401 A1 WO2021093401 A1 WO 2021093401A1 CN 2020110736 W CN2020110736 W CN 2020110736W WO 2021093401 A1 WO2021093401 A1 WO 2021093401A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- the invention belongs to the field of medicine, and specifically relates to the application of LncRNA-266 in preparing drugs for inducing the differentiation of brown adipocytes and promoting energy metabolism in the body.
- Obesity is a physiological state of the body's energy metabolism disorder, which is manifested by the accumulation of excess energy in the body in the form of fat. Obesity is a serious threat to human health. It can induce a variety of metabolic diseases, such as fatty liver, type 2 diabetes, cardiovascular disease, and certain types of cancer.
- the body's adipose tissue is mainly divided into two types, namely white adipose tissue and brown adipose tissue.
- white adipose cells In terms of energy metabolism, white adipose cells contain a large amount of triglycerides, so white adipose tissue is mainly used as an organ for storing energy; on the contrary, brown adipose cells contain a large number of mitochondria and highly express the uncoupling protein UCP1. Uncoupling converts stored energy into heat and emits it, so BAT is considered an energy-consuming organ. Therefore, it is reasonable to speculate that if the human body's BAT heat production is increased by some method, it will undoubtedly delay weight gain and reduce the development of obesity.
- LncRNAs Long noncoding RNAs
- LncRNAs Long noncoding RNAs
- the purpose of the present invention is to provide the medical use of long-chain non-coding RNA (LncRNA-266), and the specific technical solutions are as follows:
- LncRNA-266 in the preparation of drugs for inducing the differentiation of brown adipocytes, the cDNA sequence of LncRNA-266 is shown in SEQ ID No:1.
- LncRNA-266 can induce the differentiation of beige adipocytes into brown adipocytes, thereby promoting the body's energy metabolism and inhibiting weight gain.
- LncRNA-266 can lower the body's blood sugar level and improve insulin sensitivity.
- Another object of the present invention is to provide a drug for inducing adipocyte differentiation, comprising LncRNA-266 or a vector expressing LncRNA-266.
- the vector is selected from one or more of plasmids, expression cassettes, viruses, and cells.
- the medicine can induce the differentiation of beige adipocytes into brown adipocytes, consume body energy, and inhibit body weight gain.
- the medicine can lower the blood sugar level of the body and improve insulin sensitivity.
- the present invention studies the inducing effect of LncRNA-266 on the browning of beige fat.
- an adenovirus expressing LncRNA-266 (Ad-LncRNA-266)
- using the constructed adenovirus expressing LncRNA to transduce the precursor adipocytes, and then proceeding with the targeted induction of brown adipocytes, oil red staining and qRT-PCR results
- LncRNA-266 can significantly induce the differentiation of pre-adipocytes into brown adipocytes, and the brown adipocyte-specific gene UCP1 also shows a high expression trend.
- the present invention constructs its interfering virus (Ad-LncRNA-266 shRNA).
- the constructed interfering virus is used to transduce precursor adipocytes.
- LncRNA-266 can inhibit the transformation of precursor adipocytes to brown adipocytes.
- UCP1 gene expression also showed a downward trend, confirming the promotion of LncRNA-266 in the process of inducing the differentiation of pre-adipocytes into brown adipocytes.
- the adenovirus expressing LncRNA-266 (Ad-LncRNA-266) is directly injected into the inguinal beige adipose tissue of obese mice (type II diabetes model mice).
- LncRNA-266 can significantly reduce blood glucose and serum insulin levels in type 2 diabetes model mice, and significantly improve the glucose tolerance and insulin tolerance of obese mice, indicating that LncRNA-266 can improve glucose clearance in type 2 diabetes model mice. Ability, and can improve its insulin sensitivity.
- Figure 1 shows that LncRNA-266 induces the differentiation of pre-adipocytes into brown fat.
- Figure 1A shows the results of oil red staining of fat cells;
- Figure 1B shows the expression level of UCP1 gene detected by quantitative PCR. ***p ⁇ 0.001, Student's test analysis.
- Figure 2 shows that interfering with the expression of LncRNA-266 inhibits the differentiation of pre-adipocytes into brown adipocytes.
- Figure 2A shows the oil red staining of fat cells;
- Figure 2B shows the quantitative PCR to detect the expression level of UCP1 gene. **p ⁇ 0.01, Student's test analysis.
- FIG. 3 shows the effect of LncRNA-266 on blood glucose levels in type 2 diabetic mice. **p ⁇ 0.01, Student's test analysis.
- FIG. 4 shows the effect of LncRNA-266 on serum insulin levels in type diabetic mice. *p ⁇ 0.05, Student's test analysis.
- Figure 5 shows the effect of LncRNA-266 on glucose tolerance in type 2 diabetic mice. **p ⁇ 0.01, ***p ⁇ 0.001, Student's test analysis.
- Figure 6 shows the effect of LncRNA-266 on insulin tolerance in type 2 diabetic mice. *p ⁇ 0.05, ***p ⁇ 0.001, Student's test analysis.
- Figure 7 shows that LncRNA-266 treatment promotes the transformation of beige adipose tissue in the groin of mice to brown adipose tissue.
- Figure 7A is a morphological map of fat;
- Figure 7B is a quantitative PCR to detect the expression level of UCP1 gene. ***p ⁇ 0.001, Student's test analysis.
- overexpression virus was constructed using Invitrogen's Gateway series kit. First, PCR is used to amplify the complete sequence of LncRNA, and then it is connected to the intermediate vector pENTR3C-Entry Vector and sent to the company for sequencing. The sequenced vector is connected to the target vector (pAd-CMV-DEST Vector) through recombination (LR). After the sequencing detection is correct, the positive plasmids are extracted, and cut by endonuclease Pac I, and then transfected into 293A cells with Invitrogen's Lipofectamine 2000 liposomes. Observe the formation of virus plaques in about a week.
- 293A cells are collected and subjected to repeated freezing and thawing at -80°C and 37°C to break the cells. The supernatant is collected by centrifugation to obtain the first generation virus.
- the second-generation virus is obtained in the same way.
- the second-generation virus can be stored in a low temperature refrigerator at -80°C for subsequent experiments after virus titer detection.
- the control virus was an adenovirus expressing LacZ (Ad-LacZ).
- the plasmid for constructing the virus is pAd/CMV/V5-GW/LacZ provided by Invitrogen, and it is transfected into 293A cells after Pac I single-cutting. The transfection and virus preparation process are the same as above.
- Interference virus preparation Using Invitrogen's Gateway series kits, first use Invitrogen's online software to design shRNA, send it to Life to synthesize single-stranded DNA, and then anneal to form double-stranded DNA, which is connected to the intermediate vector BLOCK-it U6 RNAi Entry Vector Then it was sent to the company for sequencing. The vector that was sequenced correctly was connected to the target vector (pAd/BLOCK-iT-DEST Vector) by recombination (LR). After the sequencing and detection was correct, the positive plasmid was prepared and passed the endonuclease Pac After I cut, 293A cells were transfected with Invitrogen's Lipofectamine 2000 liposome. Virus collection and amplification are as described above. The control virus is Ad-NC shRNA, and its construction method is the same as above.
- 2ml culture medium DMEM high glucose medium, 20% fetal bovine serum, 20mM Hepes, 100U/ml penicillin/streptomycin
- DMEM high glucose medium, 20% fetal bovine serum, 20mM Hepes, 100U/ml penicillin/streptomycin
- the precursor adipocytes were added with 1 ⁇ 10 8 PFU overexpression virus (Ad-LncRNA- 266) or interference virus (Ad-LncRNA-266 shRNA) treatment, aspirate the medium after 24 hours, and change the medium for brown adipocyte induction.
- the composition of the induction medium includes DMEM high glucose medium, 10% fetal bovine serum, 20mM insulin, 1nM T3, 0.5mM IBMX, 125 ⁇ M indomethacin, 1 ⁇ M dexamethasone. Rosiglitazone was added 2 days after induction. After 2 days, change to basic induction medium (DMEM high glucose medium, 10% fetal calf serum, 20mM insulin, 1nM T3) for 3-4 days of induction, and change to fresh medium every other day.
- Trizol reagent product of Invitrogen
- SYBR Green Supermix product of Bio-Rad
- the detection instrument is iQ5 Multicolor Real-Time PCR Detection System (Product of Bio-Rad). Results 2- ⁇ Ct method calculates mRNA levels.
- the 18S housekeeping gene was used to calibrate the mRNA level.
- the primer sequences used are as follows:
- 18S rRNA forward primer 5'-AGTCCCTGCCCTTTGTACACA-3' (SEQ ID No: 2);
- 18S rRNA negative primer 5'-CGTTCCGAGGGCCTCACT-3' (SEQ ID No: 3);
- UCP1 forward primer 5’-AGGCTTCCAGTACCATTAGGT-3’ (SEQ ID No: 4);
- UCP1 negative primer 5'-CTGAGTGAGGCAAAGCTGATTT-3' (SEQ ID No: 5).
- mice 6-week-old male C57BL/6J mice were fed with a high-fat diet (45% calories from fat) for 90 days to induce obesity model mice (ie, type 2 diabetes model mice).
- the adenovirus expressing LncRNA-266 (Ad-LncRNA-266) was directly injected into the beige adipose tissue of the inguinal groin of type 2 diabetes model mice, and the dosage was 40 ⁇ l of virus (1 ⁇ 10 12 PFU) injected into the groin of each mouse. .
- the mice in the control group were injected with the same dose of control virus (Ad-LacZ). Three weeks after the virus injection, various physiological indicators were tested.
- FIG. 4 shows that LncRNA-266 can reduce serum insulin levels and improve hyperinsulinemia in type II diabetic model mice.
- mice were fasted overnight (from 8pm to 8am the next day), and D-glucose (0.5g/kg) was intraperitoneally injected into the mice.
- the tail vein blood was taken at 0, 15, 30, 60, and 90 minutes after the injection of glucose, and the blood glucose at each time point was measured with a blood glucose meter (Bayer) to investigate the glucose tolerance.
- the results are shown in FIG. 5.
- the results in Figure 5 show that LncRNA-266 treatment can significantly promote the clearance of glucose in peripheral blood in type 2 diabetes model mice.
- mice were fasted for 6 hours (from 8am to 2pm), and the mice were intraperitoneally injected with recombinant human insulin (0.75IU/kg) (purchased from Eli Lilly, Indianapolis, IN).
- recombinant human insulin (0.75IU/kg) (purchased from Eli Lilly, Indianapolis, IN).
- the tail vein blood was taken to measure the blood glucose at each time point with a blood glucose meter (Bayer) to investigate the insulin resistance test.
- the results are shown in FIG. 6.
- the results in Figure 6 show that treatment with LncRNA-266 can enhance the responsiveness of type 2 diabetes model mice to insulin and improve insulin sensitivity.
- FIG. 7A The general morphology of the groin fat tissue is shown in Figure 7A.
- the results in Figure 7A show that LncRNA-266 treatment significantly darkens the color of the inguinal adipose tissue;
- Figure 7B is a quantitative PCR test, and the results show that LncRNA-266 significantly increases the UCP1 gene expression level in the inguinal adipose tissue.
- the results in Figure 7 show that LncRNA-266 treatment promotes the transformation of beige adipose tissue to brown adipose tissue in the mouse groin.
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Abstract
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Claims (8)
- LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用,LncRNA-266的cDNA序列如SEQ ID No:1所示。
- 如权利要求1所述的应用,其特征在于所述LncRNA-266诱导米色脂肪细胞向棕色脂肪细胞分化,进而促进机体能量代谢。
- 如权利要求1所述的应用,其特征在于所述LncRNA-266抑制体重增长。
- 如权利要求1所述的应用,其特征在于所述LncRNA-266降低机体血糖水平、提高胰岛素敏感性。
- 一种诱导棕色脂肪细胞分化药物,其特征在于包含LncRNA-266或表达LncRNA-266的载体。
- 如权利要求5所述的药物,其特征在于所述载体选自质粒、表达框、病毒、细胞中的一种或几种。
- 如权利要求5所述的药物,其特征在于所述药物通过诱导米色脂肪细胞向棕色脂肪细胞分化,消耗机体能量,抑制机体体重增长。
- 如权利要求5所述的药物,其特征在于所述药物降低机体血糖水平、提高胰岛素敏感性。
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