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 PDF

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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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lncrna
differentiation
adipocytes
brown
mice
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孙诚
汤欣
马谨瑜
刘晓宇
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南通大学
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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
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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

一种长链非编码RNA(LncRNA-266)的医药用途，即LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用，能够促进机体能量消耗、抵抗肥胖。通过LncRNA-266处理肥胖小鼠，结果表明LncRNA-266可促进肥胖小鼠米色脂肪细胞向棕色脂肪细胞分化，消耗机体能量，从而抑制体重增长。进一步发现，LncRNA-266可降低肥胖小鼠血糖水平、提高胰岛素敏感性。

Description

LncRNA-266在制备诱导棕色脂肪细胞分化药物中的应用 技术领域
本发明属于医药领域，具体涉及LncRNA-266在制备诱导棕色脂肪细胞分化、促进机体能量代谢药物中的应用。
背景技术
肥胖是机体能量代谢失调的一种生理状态，表现为多余能量以脂肪的形式在体内大量积累。肥胖严重威胁着人类健康，它会诱发多种代谢性疾病，如脂肪肝、2型糖尿病、心血管疾病、特定类型的癌症等。机体的脂肪组织主要分为两类，即白色脂肪组织和棕色脂肪组织。在能量代谢方面，白色脂肪细胞内含有大量甘油三酯，因此白色脂肪组织主要作为储存机体能量的器官；与此相反，棕色脂肪细胞内含有大量线粒体，并高表达解偶联蛋白UCP1，可通过解偶联作用将储存的能量转化成热能散发出去，因此BAT被认为是消耗能量的器官。因此可以合理推测，若通过某种方法使人体BAT产热增加，无疑将可延缓体重增加，减轻肥胖症的发展。
长链非编码RNA(long noncoding RNAs,LncRNAs)是指一类长度大于200bp，没有编码蛋白功能的RNA分子。早期认为这些LncRNAs只是基因转录的副产物，没有特定的生理功能。但是，最近研究发现，很多LncRNAs具有非常重要的生理功能，其水平异常可导致发育缺陷及多种疾病的发生与发展。
发明内容
本发明的目的在于提供长链非编码RNA(LncRNA-266)的医药用途，具体技术方案如下：
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。
LncRNA-266能够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、抑制体重增长。
进一步的，LncRNA-266能够降低机体血糖水平、提高胰岛素敏感性。
本发明另一目的在于提供一种诱导脂肪细胞分化药物，包含LncRNA-266或表达LncRNA-266的载体。
所述载体选自质粒、表达框、病毒、细胞中的一种或几种。
所述药物能够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。
所述药物能够降低机体血糖水平、提高胰岛素敏感性。
本发明研究了LncRNA-266对米色脂肪棕色化的诱导作用。通过构建表达LncRNA-266的腺病毒(Ad-LncRNA-266)，用所构建好的表达LncRNA的腺病毒转导前体脂肪细胞，然后进行棕色脂肪细胞定向诱导，油红染色及qRT-PCR结果表明LncRNA-266可显著诱导前体脂肪细胞向棕色脂肪细胞分化，并且棕色脂肪细胞特异性基因UCP1也呈高表达趋势。进一步的，本发明构建了其干扰病毒(Ad-LncRNA-266 shRNA)，用构建好的干扰病毒转导前体脂肪细胞，结果发现下调LncRNA-266的表达可抑制前体脂肪细胞向棕色脂肪细胞的分化，并且UCP1基因表达也呈下降趋势，证实LncRNA-266在诱导前体脂肪细胞向棕色脂肪细胞分化过程中的促进作用。本发明在活体动物水平上，将表达LncRNA-266的腺病毒(Ad-LncRNA-266)直接注射于肥胖小鼠(Ⅱ型糖尿病模型小鼠)腹股沟米色脂肪组织。病毒注射3周后，发现腹股沟米色脂肪组织向棕色脂肪组织转变，UCP1的表达也显著升高。另外，LncRNA-266可以显著降低Ⅱ型糖尿病模型小鼠血糖及血清胰岛素水平，显著提高肥胖小鼠的葡萄糖耐受和胰岛素耐受能力，说明LncRNA-266可提高Ⅱ型糖尿病模型小鼠体内葡萄糖清除能力，并能提高其胰岛素敏感性。
附图说明
图1为LncRNA-266诱导前体脂肪细胞向棕色脂肪分化。图1A为脂肪细胞油红染色结果；图1B为定量PCR检测UCP1基因表达水平。***p<0.001，Student's t test分析。
图2为干扰LncRNA-266表达抑制前体脂肪细胞向棕色脂肪细胞分化。图2A为脂肪细胞油红染色；图2B为定量PCR检测UCP1基因表达水平。**p<0.01，Student's t test分析。
图3为LncRNA-266对Ⅱ型糖尿病小鼠血糖水平的影响。**p<0.01，Student's t test分析。
图4为LncRNA-266对型糖尿病小鼠血清胰岛素水平的影响。*p<0.05，Student's t test分析。
图5为LncRNA-266对Ⅱ型糖尿病小鼠葡萄糖耐受能力的影响。**p<0.01，***p<0.001，Student's t test分析。
图6为LncRNA-266对Ⅱ型糖尿病小鼠胰岛素耐受能力的影响。*p<0.05，***p<0.001，Student's t test分析。
图7为LncRNA-266处理促进小鼠腹股沟米色脂肪组织向棕色脂肪组织转变。图7A为脂肪形态图；图7B为定量PCR检测UCP1基因表达水平。***p<0.001，Student's t test 分析。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不受实施例限制。
在本发明中使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明，应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。
在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1 LncRNA-266过表达及干扰病毒构建
过表达病毒制备：本研究中过表达病毒采用Invitrogen公司的Gateway系列试剂盒进行构建。首先用PCR扩增出LncRNA的完整序列，然后连入中间载体pENTR3C-Entry Vector后送往公司测序，测序正确的载体通过重组的方法(LR)连入到目标载体(pAd-CMV-DEST Vector)中，测序检测正确后，将阳性质粒中抽，并经核酸内切酶Pac I单切后用Invitrogen公司的Lipofectamine 2000脂质体转染293A细胞。一周左右观察病毒斑形成情况，待绝大部分病毒斑出现后收集293A细胞并进行-80℃和37℃的反复冻融使细胞破碎，离心收集上清即获得第一代病毒，将第一代病毒继续感染293A细胞后同样方法获得第二代病毒，第二代病毒进行病毒滴度检测后可保存于-80℃低温冰箱用于后续实验。对照病毒为表达LacZ的腺病毒(Ad-LacZ)。构建该病毒的质粒为pAd/CMV/V5-GW/LacZ由Invitrogen公司提供，经Pac I单切后转染293A细胞，转染及病毒制备过程与上述相同。
干扰病毒制备：采用Invitrogen公司的Gateway系列试剂盒，首先运用Invitrogen公司在线软件设计shRNA，送往Life公司合成单链的DNA，然后退火形成双链DNA，连入中间载体BLOCK-it U6 RNAi Entry Vector后送往公司测序，测序正确的载体通过重组的方法(LR)连入到目标载体(pAd/BLOCK-iT-DEST Vector)中，测序检测正确后，制备阳性质粒，并经核酸内切酶Pac I单切后用Invitrogen公司的Lipofectamine 2000脂质体转染293A细胞。病毒收集与扩增如前所述。对照病毒为Ad-NC shRNA，其构建方法与上述相同。
实施例2 LncRNA-266诱导前体脂肪细胞分化
原代前体脂肪细胞培养：8周龄雄性C57BL/6J小鼠，取腹股沟白色脂肪组织于离心管中，胶原酶消化液(胶原酶1mg/ml溶于分离缓冲液-----包括0.123M NaCl,5mM KCl,1.3mM CaCl 2,5nM葡萄糖,100mM Hepes,4％BSA)37℃水浴锅消化30min，间隔5min振荡一次，100μm尼龙筛网过滤，离心，200g×5min。去上清液，加入2ml分离缓冲液充分悬浮细胞，离心， 200g×5min。去上清液，加入2ml培养液(DMEM高糖培养基，20％胎牛血清，20mM Hepes,100U/ml青霉素/链霉素)充分悬浮细胞，种于培养皿中，置于37℃，5％CO 2培养箱中培养。
棕色脂肪细胞定向诱导分化：为检测LncRNA-266对脂肪前体细胞向棕色脂肪细胞的转化，在加诱导培养基之前，前体脂肪细胞分别加入1×10 8PFU过表达病毒(Ad-LncRNA-266)或干扰病毒(Ad-LncRNA-266 shRNA)处理，24小时后吸取培养基，换棕色脂肪细胞诱导培养基。诱导培养基组成包括DMEM高糖培养基，10％胎牛血清，20mM胰岛素，1nM T3，0.5mM IBMX，125μM吲哚美辛，1μM地塞米松。诱导2天后加入罗格列酮。2天后换用基础诱导培养基(DMEM高糖培养基，10％胎牛血清，20mM胰岛素，1nM T3)诱导3-4天，其间隔天换新鲜培养基。
(1)油红染色
细胞处理结束后，去除上清培养基，加PBS清洗一次，移除PBS，加4％多聚甲醛固定，室温20-30min。随后，用PBS清洗1-2次，彻底移除PBS，加入油红工作液，将细胞置于低速摇床上孵育50-60min(室温)，移除油红工作液，加下PBS清洗3次，倒置显微镜拍照，结果如图1A和2A所示，图1A结果表明LncRNA-266可显著增加脂滴在细胞中的积累，说明前体脂肪细胞向棕色脂肪细胞的诱导分化。图2A显示下调LncRNA-266的表达则减少脂滴在细胞中的积累，说明抑制了前体脂肪细胞向棕色脂肪细胞的诱导分化。
(2)定量PCR检测基因表达水平
使用Trizol试剂(Invitrogen公司产品)提取细胞总RNA，再经cDNA合成试剂盒(Bio-Rad公司产品)转录成cDNA。以此cDNA为模板，用SYBR Green Supermix(Bio-Rad公司产品)分析基因表达水平。检测仪器为iQ5 Multicolor Real-Time PCR Detection System(Bio-Rad公司产品)。结果2-ΔCt方法计算mRNA水平。以18S持家基因来校准mRNA的水平。所使用的引物序列如下：
18S rRNA正向引物:5’-AGTCCCTGCCCTTTGTACACA-3’(SEQ ID No：2)；
18S rRNA负向引物:5’-CGTTCCGAGGGCCTCACT-3’(SEQ ID No：3)；
UCP1正向引物:5’-AGGCTTCCAGTACCATTAGGT-3’(SEQ ID No：4)；
UCP1负向引物:5’-CTGAGTGAGGCAAAGCTGATTT-3’(SEQ ID No：5)。
qRT-PCR结果如图1B和2B所示，图1B结果表明LncRNA-266可显著诱导棕色脂肪细胞特异性基因UCP1表达增高，图2B结果表明下调LncRNA-266的表达可显著下调UCP1基因表达。这些结果提示LncRNA-266可显著诱导前体脂肪细胞向棕色脂肪细胞分化。
实施例3 LncRNA-266对Ⅱ型糖尿病小鼠的糖代谢的影响
6周龄雄性C57BL/6J小鼠，用高脂饮食(Research Diet,45％calories from fat)饲喂90天，诱导成肥胖模型小鼠(即Ⅱ型糖尿病模型小鼠)。将表达LncRNA-266的腺病毒(Ad-LncRNA-266)直接注射于Ⅱ型糖尿病模型小鼠腹股沟米色脂肪组织，使用剂量为每只小鼠单侧腹股沟处注射40μl病毒(1×10 12PFU)。对照组小鼠注射同等剂量的对照病毒(Ad-LacZ)。病毒注射3周后，检测各项生理指标检测。
小鼠禁食12小时后，采用尾静脉取血，血糖水平用血糖仪(Bayer,Mishawaka,IN)定量检测血糖水平，结果如图3所示。图3结果表明，LncRNA-266处理可降低Ⅱ型糖尿病模型小鼠的血糖水平。
小鼠禁食12小时后，采用尾静脉取血，使用小鼠血清胰岛素水平用试剂盒(Rat/mouse insulin ELISA kit,Mercodia,2758702)测定血清胰岛素水平，结果如图4所示。图4结果表明，LncRNA-266可降低血清胰岛素水平，改善型糖尿病模Ⅱ型小鼠的高胰岛素血症。
小鼠过夜禁食(从8pm到次日8am)，给小鼠腹腔注射D-葡萄糖(0.5g/kg)。分别在注射葡萄糖后的0，15，30，60，90min取尾静脉血用血糖仪(Bayer)测定各时间点的血糖，考察葡萄糖耐受能力，结果如图5所示。图5结果表明，LncRNA-266处理可显著促进Ⅱ型糖尿病模型小鼠对外周血中葡萄糖的清除能力。
小鼠禁食6小时(从8am到2pm)，给小鼠腹腔注射重组人胰岛素(0.75IU/kg)(购于Eli Lilly，Indianapolis，IN)。分别在注射葡萄糖后的0，15，30，60，90min取尾静脉血用血糖仪(Bayer)测定各时间点的血糖，考察胰岛素耐受能力检测，结果如图6所示。图6结果表明，LncRNA-266处理可增强Ⅱ型糖尿病模型小鼠对胰岛素的响应能力，提高胰岛素敏感性。
实验结束，小鼠经麻醉后处死，快速解剖小鼠，提取腹股沟脂肪用于下述检测。腹股脂肪组织的大体形态图如图7A所示。图7A结果表明LncRNA-266处理使腹股沟脂肪组织的颜色明显加深；图7B为定量PCR检测，结果表明LncRNA-266显著提高了腹股脂肪组织中UCP1基因表达水平。综上，图7结果显示LncRNA-266处理促进小鼠腹股沟米色脂肪组织向棕色脂肪组织转变。

Claims (8)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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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