WO2019019934A1 - Tm9sf1基因作为靶点在血管性疾病中的应用 - Google Patents

Tm9sf1基因作为靶点在血管性疾病中的应用 Download PDF

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WO2019019934A1
WO2019019934A1 PCT/CN2018/095821 CN2018095821W WO2019019934A1 WO 2019019934 A1 WO2019019934 A1 WO 2019019934A1 CN 2018095821 W CN2018095821 W CN 2018095821W WO 2019019934 A1 WO2019019934 A1 WO 2019019934A1
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tm9sf1
gene
candidate agent
expression
tm9sf1 gene
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肖娟
黄艳丽
毛春
杨林
殷焦
何小明
沈小芳
邓文彬
吴云涛
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湖北文理学院
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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Definitions

  • the present disclosure relates to the field of biotechnology, and in particular to the use of the TM9SF1 gene as a target in vascular diseases.
  • Endothelial cells are monolayers located on the inner wall of blood vessels and play a key role in the normal function of blood vessels. Their dysfunction is often closely related to vascular diseases.
  • the nine-transmembrane superfamily protein member 1 (TM9SF1) is an evolutionarily conserved nine-time transmembrane protein expressed in human tissues and various cell lines.
  • the TM9SF1 protein is expressed by the TM9SF1 gene, and studies on the TM9SF1 protein and the TM9SF1 gene, especially functional studies, are still rare. As for the study between endothelial cell function and the TM9SF1 gene, it is rare to report it.
  • a first object of the present disclosure includes, but is not limited to, the use of providing a TM9SF1 gene for screening for a medicament for treating or inhibiting vascular endothelial cell inflammation.
  • a second object of the present disclosure includes, but is not limited to, the use of the TM9SF1 gene for screening drugs for inhibiting tumor tissue angiogenesis.
  • a third object of the present disclosure includes, but is not limited to, the use of the TM9SF1 gene for screening drugs for the treatment of hypertension.
  • a fourth object of the present disclosure includes, but is not limited to, the use of an agent that provides inhibition or silencing of the expression of the TM9SF1 gene in the preparation of a drug associated with vascular endothelial cell inflammation, angiogenesis, hypertension.
  • a fifth object of the present disclosure includes, but is not limited to, providing a drug for treating or inhibiting vascular endothelial cell inflammation, for inhibiting tumor tissue angiogenesis, or for treating hypertension.
  • a sixth object of the present disclosure includes, but is not limited to, providing a method of treating or inhibiting vascular endothelial cell inflammation, a method of inhibiting tumor tissue angiogenesis, or a method of treating hypertension.
  • the TM9SF1 gene is used as a target for screening for drugs for treating or inhibiting inflammation of vascular endothelial cells.
  • the TM9SF1 gene is used as a target for screening drugs for inhibiting tumor tissue angiogenesis.
  • the TM9SF1 gene is used as a target for screening drugs for the treatment of hypertension.
  • the medicament contains an agent that inhibits or silences expression of the TM9SF1 gene and a pharmaceutically acceptable excipient.
  • a method of treating or inhibiting inflammation of vascular endothelial cells, a method of inhibiting angiogenesis of tumor tissue, or a method of treating hypertension comprising: administering a drug as described above to a subject.
  • the present invention uses human umbilical vein endothelial cells (HUVEC) as a research object, and uses RNA interference strategy to find that two important genes related to HUVEC inflammation are IL1 ⁇ , IL8 and vasoconstriction after interfering with endogenous TM9SF1 gene.
  • the expression of the related gene ACE1 was down-regulated, suggesting that the expression of TM9SF1 gene and IL1 ⁇ , IL8 and ACE1 genes are positively regulated.
  • TM9SF1 By inhibiting or silencing the expression of TM9SF1 gene, the expression of IL1 ⁇ , IL8 and ACE1 genes can be inhibited or silenced.
  • Therapeutic or prophylactic purposes for vascular diseases associated with the expression levels of the IL1 ⁇ , IL8 and ACE1 genes can be achieved.
  • the TM9SF1 gene can be used as a new target for screening drugs for treating or inhibiting vascular endothelial cell inflammation (associated with IL1 ⁇ gene expression), for inhibiting tumor tissue angiogenesis (with IL8 gene expression).
  • Related drugs in the field of drugs for the treatment of hypertension (associated with ACE1 gene expression);
  • an agent that inhibits or silences the expression of the TM9SF1 gene can be used in the fields of preparing a drug for treating or inhibiting vascular endothelial cell inflammation, a drug for inhibiting tumor tissue angiogenesis, and a drug for treating hypertension.
  • TM9SF1 gene provided by the present disclosure as a target in vascular diseases provides a new idea and means for treating and preventing vascular diseases.
  • Figure 3 is a graph showing the relative expression levels of inflammatory genes IL1, IL8 and ACE1 in HUVECs following transfection of specific siRNAs according to an embodiment of the present disclosure.
  • TM9SF1 gene provided by the examples of the present disclosure as a target in vascular diseases will be specifically described below.
  • the TM9SF1 gene was first cloned in 1997. As of now, there are very few reports on this gene, and the expression research is the main one. The literature on its functional research is currently searchable, and it has been reported that it can induce HeLa cells to occur. Phago, but did not reveal the relevant mechanisms. The biological function of the TM9SF1 gene in cells other than HeLa has not been reported.
  • RNA interference technology is a molecular biology technology widely used in recent years, and it is of great significance in exploring gene function and gene therapy. Compared with another strategy gene overexpression commonly used to study gene function, RNA interference can better reflect the true physiological state of the body. siRNA is the most commonly used tool for RNA interference, and it has the advantages of high efficiency and specificity.
  • the present invention uses human umbilical vein endothelial cells (HUVEC) as a research object, and uses RNA interference strategy to find that two important genes related to HUVEC inflammation are IL1 ⁇ , IL8 and vasoconstriction after interfering with endogenous TM9SF1 gene.
  • the expression of the related gene ACE1 was down-regulated, suggesting that the expression of TM9SF1 gene and IL1 ⁇ , IL8 and ACE1 genes are positively regulated.
  • TM9SF1 By inhibiting or silencing the expression of TM9SF1 gene, the expression of IL1 ⁇ , IL8 and ACE1 genes can be inhibited or silenced.
  • Therapeutic or prophylactic purposes for vascular diseases associated with the expression levels of the IL1 ⁇ , IL8 and ACE1 genes can be achieved.
  • the present disclosure provides the use of the TM9SF1 gene as a target in the following aspects.
  • the present disclosure provides the use of the TM9SF1 gene as a target for screening drugs for treating or inhibiting vascular endothelial cell inflammation.
  • the above drug targets the TM9SF1 gene, and inhibits or silences the expression of the TM9SF1 gene.
  • vascular endothelial cells are located between plasma and vascular tissue. They not only complete the metabolic exchange of plasma and tissue fluid, but also synthesize and secrete a variety of biologically active substances to ensure normal contraction and relaxation of blood vessels. It is known that endothelial cells are an inflammatory cell, and its inflammatory state has an important influence on the pathophysiological processes of various diseases such as atherosclerosis, aneurysm and diabetic angiopathy. Endothelial cells can produce a variety of proinflammatory cytokines during inflammation, which plays a crucial role in the further development of inflammation. For example, extracellular IL1 ⁇ can activate endothelial cells (NHEK S, CLANCY R, LEE K A, et al.
  • endothelial cells can also produce IL1 ⁇ after some stimulation, and play an important role in endothelial cell inflammatory response (XIA X, SHI Q, SONG) X, et al.Tetrachlorobenzoquinone Stimulates NLRP3Inflammasome-Mediated Post-Translationa Activation and Secretion of IL-1beta in the HUVEC Endothelial Cell Line [J]. Chem Res Toxicol, 2016, 29(3):421-429), therefore trying to inhibit the expression of IL1 ⁇ in endothelial cells is important for inhibiting endothelial cell inflammatory response The meaning.
  • TM9SF1 may play a positive regulatory role in endothelial cell inflammation.
  • the TM9SF1 gene can be used as a target for screening drugs for treating or inhibiting vascular endothelial cell inflammation.
  • the selected drug targets the TM9SF1 gene, inhibits or silences the expression of the TM9SF1 gene, indirectly inhibits the expression level of the IL1 ⁇ gene, and functions to treat or inhibit vascular endothelial cell inflammation.
  • the biological sample containing the TM9SF1 gene is cultured in the presence of a candidate agent
  • the biological sample containing the TM9SF1 gene is cultured in the absence of the candidate agent;
  • IL1 ⁇ expression level of the above biological sample in the presence of the candidate agent and in the absence of the candidate agent, wherein the IL1 ⁇ expression level is lower than the absence of the candidate agent in the presence of the candidate agent.
  • the lower level of IL1 ⁇ expression is an indication of the candidate agent as a drug for treating or inhibiting inflammation of vascular endothelial cells.
  • the candidate reagent targets the TM9SF1 gene and inhibits or silences its expression level.
  • the candidate agent may be an siRNA against the TM9SF1 gene; or may be an antibody against the TM9SF1 protein, which inhibits the activity or amount of the TM9SF1 protein at the protein level.
  • the biological sample may be a human umbilical vein endothelial cell or a mouse umbilical vein endothelial cell.
  • the present disclosure provides the use of an agent that inhibits or silences expression of a TM9SF1 gene for the preparation of a medicament for treating or inhibiting vascular endothelial cell inflammation.
  • an agent that inhibits or silences the expression of the TM9SF1 gene can be used as a new use in the preparation of a medicament for treating or inhibiting inflammation of vascular endothelial cells. It provides a new idea and means for treating or inhibiting vascular endothelial cell inflammation.
  • the above reagent is an siRNA of the TM9SF1 gene.
  • the two "TT" bases at the 3' end are used to increase siRNA stability, and positions 1-19 are used to interact with the target gene.
  • the siRNA having the sequence of SEQ ID NO. 1 was able to effectively inhibit the expression of TM9SF1 gene, and the relative expression level of TM9SF1 gene was (0.11 ⁇ 0.04), P ⁇ 0.005), which was much lower than that of the control group. Its interference efficiency is greater than 50%. At the same time, the expression level of IL1 ⁇ gene was (0.30 ⁇ 0.09, (P ⁇ 0.001)), which was significantly lower than that of the control group.
  • This siRNA has high interference efficiency and can also be used as a brand new drug for treating or inhibiting vascular endothelial cell inflammation.
  • the present disclosure provides the use of the TM9SF1 gene as a target for screening drugs for inhibiting tumor tissue angiogenesis.
  • the above drug targets the TM9SF1 gene, and inhibits or silences the expression of the TM9SF1 gene.
  • endothelial cells which itself is also one of the important participants in endothelial cell inflammation (BORGES L E, BLOISE E, DELA C C, et al. Urocortin 1expression and secretion by human umbilical vein endothelial cells: In Vitro effects of interleukin 8,interferon gamma,lipopolysaccharide,endothelin 1,prostaglandin F-2alpha,estradiol,progesterone and dexamethasone[J].Peptides,2015,74:64-69.),Transplantation of endothelial cells (JU L, ZHOU Z , JIANG B, et al.
  • the present study found that the expression level of IL8 in HUVEC was significantly decreased after interfering with endogenous TM9SF1. From the reverse side, TM9SF1 could promote the expression of IL8 in HUVEC cells. Therefore, the TM9SF1 gene can be used as a target for screening drugs for inhibiting tumor tissue angiogenesis.
  • the selected drug targets the TM9SF1 gene, inhibits or silences the expression of the TM9SF1 gene, indirectly inhibits the expression level of the IL8 gene, and inhibits tumor tissue angiogenesis.
  • the application includes:
  • the biological sample containing the TM9SF1 gene is cultured in the presence of a candidate agent
  • the biological sample containing the TM9SF1 gene is cultured in the absence of the candidate agent;
  • IL8 expression level of the above biological sample in the presence of the candidate agent and in the absence of the candidate agent, wherein, in the presence of the candidate agent, the IL8 expression level is lower than the absence of the candidate agent
  • the lower IL8 expression level is an indication of the candidate agent as a drug that inhibits tumor tissue angiogenesis.
  • the candidate reagent targets the TM9SF1 gene and inhibits or silences its expression level.
  • the cDNA sequence of the TM9SF1 gene is shown in SEQ ID NO.
  • the candidate agent may be an siRNA against the TM9SF1 gene; or may be an antibody against the TM9SF1 protein, which inhibits the activity or amount of the TM9SF1 protein at the protein level.
  • the biological sample may be a human umbilical vein endothelial cell or a mouse umbilical vein endothelial cell.
  • the present disclosure provides the use of an agent that inhibits or silences expression of a TM9SF1 gene for the preparation of a medicament for inhibiting tumor tissue angiogenesis.
  • an agent that inhibits or silences the expression of the TM9SF1 gene can be used in the preparation of a medicament for inhibiting tumor tissue angiogenesis.
  • an agent that inhibits or silences the expression of TM9SF1 gene provides a new idea and means for inhibiting tumor tissue angiogenesis.
  • the above reagent is an siRNA of the TM9SF1 gene.
  • the siRNA having the sequence of SEQ ID NO. 1 was able to effectively inhibit the expression of TM9SF1 gene, and the relative expression level of TM9SF1 gene was (0.11 ⁇ 0.04), P ⁇ 0.005), which was much lower than that of the control group. Its interference efficiency is greater than 50%. At the same time, the expression level of IL8 gene was ((0.23 ⁇ 0.17, (P ⁇ 0.005)), which was significantly lower than that of the control group. This indicates that the siRNA has high interference efficiency and can be used as a brand new one. A drug that inhibits tumor tissue angiogenesis.
  • the present disclosure provides the use of the TM9SF1 gene as a target for screening drugs for treating hypertension.
  • the above drug targets the TM9SF1 gene, and inhibits or silences the expression of the TM9SF1 gene.
  • ACE1 also known as CD143
  • ACE1 angiotensin-converting enzyme inhibitor
  • Angiotensin-Converting Enzyme Inhibitor Prevents the Worsening of Renal Function in the Late Phase after Percutaneous Coronary Intervention [J].J Atheroscler Thromb, 2016, 23(2): 233-240.SHIH C J,CHEN H T,CHAO P W,et al.Angiotensin-converting enzyme inhibitors,angiotensin II receptor blockers and the risk of major adverse cardiac events in patients with diabetes and prior stroke:a nationwide study[J ].J Hypertens, 2016, 34(3): 567-574, 575.).
  • the TM9SF1 gene can be used as a target for screening drugs for treating hypertension.
  • the selected drug targets the TM9SF1 gene, inhibits or silences the expression of the TM9SF1 gene, and indirectly inhibits the expression level of the ACE1 gene, thereby reducing blood pressure and treating blood pressure.
  • the application includes:
  • the biological sample containing the TM9SF1 gene is cultured in the presence of a candidate agent
  • the biological sample containing the TM9SF1 gene is cultured in the absence of the candidate agent;
  • ACE1 expression level of the above biological sample in the presence of the candidate agent and in the absence of the candidate agent, wherein the ACE1 expression level is lower than the absence of the candidate agent in the presence of the candidate agent.
  • the lower ACE1 expression level is an indication of the candidate agent as a blood pressure lowering drug.
  • the candidate reagent targets the TM9SF1 gene and inhibits or silences its expression level.
  • the candidate agent may be an siRNA against the TM9SF1 gene; or may be an antibody against the TM9SF1 protein, which inhibits the activity or amount of the TM9SF1 protein at the protein level.
  • the biological sample may be a human umbilical vein endothelial cell or a mouse umbilical vein endothelial cell.
  • the present disclosure provides the use of an agent that inhibits or silences expression of a TM9SF1 gene for the preparation of a medicament for the treatment of hypertension.
  • an agent that inhibits or silences the expression of the TM9SF1 gene can be used in the preparation of a medicament for treating hypertension.
  • the agent that inhibits or silences the expression of TM9SF1 gene provides a new idea and means for treating hypertension.
  • the reagent is an siRNA of the TM9SF1 gene.
  • the siRNA having the sequence of SEQ ID NO. 1 was able to effectively inhibit the expression of TM9SF1 gene, and the relative expression level of TM9SF1 gene was (0.11 ⁇ 0.04), P ⁇ 0.005), which was much lower than that of the control group. Its interference efficiency is greater than 50%. At the same time, the expression level of ACE1 gene was (0.07 ⁇ 0.01, (P ⁇ 0.001)), which was significantly lower than that of the control group. This siRNA has high interference efficiency and can also be used as a brand new drug for lowering blood pressure.
  • the present disclosure also provides a drug having one or more of the following uses:
  • the medicament contains an agent that inhibits or silences expression of the TM9SF1 gene and a pharmaceutically acceptable excipient.
  • the above reagent is an siRNA against the TM9SF1 gene; or, the above reagent is an antibody against the TM9SF1 protein, which inhibits the activity or amount of the TM9SF1 protein at the protein level.
  • the present disclosure also provides a method for treating or inhibiting vascular endothelial cell inflammation, a method for inhibiting tumor tissue angiogenesis, or a method for treating hypertension, It comprises administering to the subject a drug provided in the sixth aspect above.
  • the subject is a mouse, a monkey, or a human.
  • the novel use of the TM9SF1 gene provided by the present disclosure as a target and a reagent for inhibiting the TM9SF1 gene in vascular diseases provides a new idea and means for treating and preventing vascular diseases.
  • HUVEC was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • TM9SF1-specific siRNA (SEQ ID NO. 1) was designed and synthesized by Jima.
  • Endogenous cell-specific medium EGM was produced by LONZA, Switzerland; trypsin (including EDTA), fetal bovine serum (FBS), phosphate buffer (PBS), etc. were produced by Hyclone, USA; transfection reagent Lipofectamine 3000 was purchased from Thermo Company, USA.
  • CD31 antibody was purchased from Immunoway, USA; immunocytochemical colorimetric kit was purchased from Beijing Zhongshang Jinqiao Co., Ltd.; CYBR Green Mix was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; CCK8 was purchased from Yusheng Company.
  • HUVEC was cultured in EGM, a special medium for endothelial cells, cultured in an incubator at 37.5 ° C, 5% CO 2 , and saturated humidity, and changed every other day.
  • EGM a special medium for endothelial cells
  • HUVEC cells When the HUVEC cells were confluent to 80%, they were fixed with 4% paraformaldehyde for 30 min, washed with PBS and perforated with 0.1% Triton X-100 for 30 min. After 30 min of goat serum blocking, rabbit anti-CD31 (Immunoway, YT0752, 1:200 dilution) was incubated overnight at 4 °C. After washing with PBS, the secondary anti-working solution was incubated at 37 ° C for 30 min, washed thoroughly with PBS, DAB was developed for 1 min, hematoxylin was counterstained for 1 min, tap water returned to blue, and neutral gum was sealed. Photographed under an orthoscopic microscope, CD31 was mainly located on the surface of the cell membrane, and the positive cells were stained with brown.
  • the cells were seeded into 6-well plates (5 ⁇ 10 5 cells/well), and after the cells were attached to the second day, they were divided into a negative control group and an interference group for transfection.
  • the transfection process of Lipofectamin 3000 was as follows: 2.5 ⁇ L Lipofectamin 3000 was added to 50 ⁇ L PBS and diluted and mixed. 2.5 ⁇ L of siRNA (SEQ ID NO. 1) (20 ⁇ M) was added to 50 ⁇ L of PBS and mixed. The two dilutions were gently mixed and allowed to stand at room temperature. 5 min, then gently drip the mixture into the medium and mix. Change the fluid 4 to 6 hours after transfection.
  • TM9SF1-specific siRNA SEQ ID NO.1
  • the cells were lysed with Trizol at 48 h, total RNA was extracted, and 1-3 ⁇ g was reverse-transcribed into cDNA, which was used as a template and detected by real-time quantitative PCR using SBRY Green dye method.
  • the level of expression of related genes qPCR amplification conditions: 95 ° C for 10 min, 95 ° C for 15 s, 60 ° C for 1 min, a total of 40 cycles. GAPDH was used as an internal reference.
  • the upstream and downstream primer sequences are as follows:
  • the data were analyzed by GraphPad Prism 5 software. The measurement data were expressed as mean ⁇ standard deviation. The unpaired t-test was used for comparison between the two groups. The difference was statistically significant at P ⁇ 0.05.
  • Figure 1 Under the phase contrast microscope, the growth state of HUVEC was good, as shown in Figure 1.
  • Figure 1 (Figure: A is the growth of HUVEC under phase contrast microscope; B is the expression of CD31 by immunocytochemistry, the strong positive cells are indicated by arrows) It is polygonal and has a few protrusions (Figure 1-A). Since CD31 is a marker molecule of endothelial cells, mainly expressed in the cell membrane, the expression of CD31 in HUVEC cells was detected by immunocytochemistry. The results showed that almost all cells were brown, but the staining depth was different, suggesting that most of the cells expressed CD31. Positive, typical endothelial cells (Fig. 1-B).
  • TM9SF1 gene after transfection of TM9SF1-specific siRNA was detected by qPCR.
  • the results are shown in Figure 2.
  • Si-NC is a negative control group and si-TM9SF1 is a transfected siRNA.
  • the interference group ** indicates P ⁇ 0.005).
  • the negative control group was used as the reference standard 1.
  • the relative expression of TM9SF1 gene in si-TM9SF1 group was (0.11 ⁇ 0.04), P ⁇ 0.005), and the interference efficiency was greater than 50%, indicating that the siRNA was siRNA. effective.
  • the present disclosure utilizes TM9SF1 specific siRNA to interfere with the endogenous expression of the gene, and the interference effect was verified by real-time quantitative PCR technology, and the expressions of IL1, IL8 and ACE1 which are closely related to endothelial cell function were significantly decreased (P ⁇ 0.005).
  • the above results suggest that the TM9SF1 gene may have an important regulatory effect on endothelial cell function.
  • TM9SF1 gene can be used as a new target for screening drugs for the treatment or inhibition of vascular endothelial cell inflammation (associated with IL1 ⁇ gene expression) for inhibiting tumor tissue angiogenesis (with IL8).
  • Gene expression-related drugs in the field of drugs for the treatment of hypertension (associated with ACE1 gene expression);
  • an agent that inhibits or silences expression of the TM9SF1 gene (for example, the siRNA represented by SEQ ID NO. 1) can be used for the preparation of a medicament for treating or inhibiting vascular endothelial cell inflammation, a medicament for inhibiting tumor tissue angiogenesis, and In the field of drugs for the treatment of hypertension.
  • the TM9SF1 gene can be used as a new target for vascular diseases, providing a new idea and means for the treatment and prevention of vascular diseases.
  • the present disclosure reveals that inhibition or silencing of the expression of the TM9SF1 gene can inhibit or silence the expression of the IL1 ⁇ , IL8 and ACE1 genes, and thus, using this finding, the TM9SF1 gene can be used as a target for screening therapy or inhibition.
  • agents that inhibit or silence TM9SF1 gene expression can also be used to prepare for the treatment or inhibition of vascular endothelial cell inflammation. Drugs, drugs that inhibit tumor tissue angiogenesis, and drugs that treat high blood pressure.

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Abstract

本公开公开了TM9SF1基因作为靶点在血管性疾病中的应用，涉及生物技术领域。本公开利用RNA干扰策略发现干扰内源性TM9SF1基因后，与HUVEC炎症相关的两个重要基因IL1β、IL8和与血管收缩密切相关的基因ACE1表达明显下调，提示TM9SF1基因与IL1β、IL8以及ACE1基因的表达具有正向调节作用，通过抑制或沉默TM9SF1基因的表达，进而可抑制或沉默IL1β、IL8以及ACE1基因的表达，进而可实现对与IL1β、IL8以及ACE1基因的表达水平相关的血管性疾病的治疗或预防目的。

Description

TM9SF1基因作为靶点在血管性疾病中的应用
本申请要求于2017年07月26日提交中国专利局的申请号为201710622579.9、名称为“TM9SF1基因作为靶点在血管性疾病中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物技术领域，具体而言，涉及TM9SF1基因作为靶点在血管性疾病中的应用。
背景技术
内皮细胞是位于血管内壁的单层细胞，对血管正常功能的发挥起着关键作用，其功能失调往往与血管性疾病密切相关。
九次跨膜超家族蛋白1(TM9SF1，transmembrane 9superfamily protein member 1)是进化保守的九次跨膜蛋白，在人体组织和多种细胞系中表达。TM9SF1蛋白由TM9SF1基因表达，目前关于TM9SF1蛋白和TM9SF1基因的研究尤其是功能研究还很少。至于，内皮细胞功能与TM9SF1基因之间的研究更是罕见有报道。
发明内容
本公开的第一目的包括但不限于提供TM9SF1基因在筛选用于治疗或抑制血管内皮细胞炎症的药物中的应用。
本公开的第二目的包括但不限于提供TM9SF1基因在筛选用于抑制肿瘤组织血管生成的药物中的应用。
本公开的第三目的包括但不限于提供TM9SF1基因在筛选用于治疗高血压的药物中的应用。
本公开的第四目的包括但不限于提供抑制或沉默TM9SF1基因表达的试剂在制备与血管内皮细胞炎症、血管生成、高血压相关药物中的应用。
本公开的第五目的包括但不限于提供一种药物，用于治疗或抑制血管内皮细胞炎症，用于抑制肿瘤组织血管生成，或用于治疗高血压。
本公开的第六目的包括但不限于提供一种方法，该方法为治疗或抑制血管内皮细胞炎症的方法，抑制肿瘤组织血管生成的方法，或者是治疗高血压的方法。
本公开是这样实现的：
TM9SF1基因作为靶点在筛选用于治疗或抑制血管内皮细胞炎症的药物中的应用。
抑制或沉默TM9SF1基因表达的试剂在制备用于治疗或抑制血管内皮细胞炎症的药物中的应用。
TM9SF1基因作为靶点在筛选用于抑制肿瘤组织血管生成的药物中的应用。
抑制或沉默TM9SF1基因表达的试剂在制备用于抑制肿瘤组织血管生成的药物中的应用。
TM9SF1基因作为靶点在筛选用于治疗高血压的药物中的应用。
抑制或沉默TM9SF1基因表达的试剂在制备用于治疗高血压的药物中的应用。
一种药物，该药物具有以下用途中一种或几种：
(1)用于治疗或抑制血管内皮细胞炎症；
(2)用于抑制肿瘤组织血管生成；
(3)用于治疗高血压；
所述药物含有抑制或沉默TM9SF1基因表达的试剂以及药学上可接受的辅料。
一种方法，该方法是治疗或抑制血管内皮细胞炎症的方法、抑制肿瘤组织血管生成的方法、或者是治疗高血压的方法，该方法包括：给予主体施用如上所述的药物。
本公开的有益效果包括：
本公开以脐静脉内皮细胞(human umbilical vein endothelial cell，HUVEC)作为研究对象，利用RNA干扰策略发现干扰内源性TM9SF1基因后，与HUVEC炎症相关的两个重要基因IL1β、IL8和与血管收缩密切相关的基因ACE1表达明显下调，提示TM9SF1基因与IL1β、IL8以及ACE1基因的表达具有正向调节作用，通过抑制或沉默TM9SF1基因的表达，进而可抑制或沉默IL1β、IL8以及ACE1基因的表达，进而可实现对与IL1β、IL8以及ACE1基因的表达水平相关的血管性疾病的治疗或预防目的。
基于此，TM9SF1基因可以作为一种新的靶点，将其应用于筛选用于治疗或抑制血管内皮细胞炎症(与IL1β基因表达相关)的药物、用于抑制肿瘤组织血管生成(与IL8基因表达相关)的药物、用于治疗高血压(与ACE1基因表达相关)的药物等领域中；
此外，抑制或沉默TM9SF1基因表达的试剂可用于制备用于治疗或抑制血管内皮细胞炎症的药物、用于抑制肿瘤组织血管生成的药物以及用于治疗高血压的药物等领域中。
本公开提供的TM9SF1基因作为靶点在血管性疾病方面的新用途，为治疗和预防血管性疾病提供了一种新的思路和手段。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例提供的HUVEC细胞培养鉴定结果；
图2为本公开实施例提供的TM9SF1特异性siRNA扰效果验证结果；
图3为本公开实施例提供的转染特异性siRNA后的HUVEC中炎症基因IL1、IL8和ACE1的相对表达量结果。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将对本公开实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
下面对本公开实施例提供的TM9SF1基因作为靶点在血管性疾病中的应用进行具体说明。
TM9SF1基因于1997年被初次克隆，截至目前关于该基因的报道非常少，且以表达性研究为主，关于其功能研究的文献目前能搜索的文献很少，有报道其可以诱导HeLa细胞发生自噬，然而并未揭示相关机制。TM9SF1基因对除HeLa以外其它细胞的生物学功能尚无报道。
RNA干扰技术是近年来得到广泛应用的分子生物学技术，其在探索基因功能和基因治疗方面具有重要意义。相比较于另一种研究基因功能常用的策略基因过表达而言，RNA干扰更能反映机体真实的生理状态。siRNA是实现RNA干扰最常用的工具，它具有高效性和特异性等优点。
本公开以脐静脉内皮细胞(human umbilical vein endothelial cell，HUVEC)作为研究对象，利用RNA干扰策略发现干扰内源性TM9SF1基因后，与HUVEC炎症相关的两个重要基因IL1β、IL8和与血管收缩密切相关的基因ACE1表达明显下调，提示TM9SF1基因与IL1β、IL8以及ACE1基因的表达具有正向调节作用，通过抑制或沉默TM9SF1基因的表达，进而可抑制或沉默IL1β、IL8以及ACE1基因的表达，进而可实现对与IL1β、IL8以及ACE1基因的表达水平相关的血管性疾病的治疗或预防目的。
基于此，本公开提供了TM9SF1基因作为靶点在以下方面中的应用。
第一方面，本公开提供了TM9SF1基因作为靶点在筛选用于治疗或抑制血管内皮细胞炎症的药物中的应用。
进一步地，上述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
血管内皮细胞位于血浆与血管组织之间，它不仅能完成血浆和组织液的代谢交换，并且能合成和分泌多种生物活性物质，以保证血管正常的收缩和舒张。目前已知内皮细胞是一种炎症细胞，其炎症状态对多种疾病的病理生理过程如动脉粥样硬化、动脉瘤和糖尿病血管病变等发生发展具有重要影响。在炎症过程中内皮细胞可以产生多种致炎细胞因子，对炎症进一步发展起到至关重要的作用。例如，细胞外的IL1β可以活化内皮细胞(NHEK S,CLANCY R,LEE K A,et al.Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1beta Pathway in Systemic Lupus Erythematosus[J].Arterioscler Thromb Vasc Biol,2017,37(4):707-716.NYMO S,GUSTAVSEN A,NILSSON P H,et al.Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1beta Secondarily to Activation of C5and CD14in Whole Blood[J].J Immunol,2016,196(5):2293-2299.Du L,DONG F,GUO L,et al.Interleukin-1beta increases permeability and upregulates the expression of vascular endothelial-cadherin in human renal glomerular endothelial cells[J].Mol Med Rep,2015,11(5):3708-3714.)，内皮细胞受到某些刺激以后也可以产生IL1β，在内皮细胞炎症应答过程中发挥重要作用(XIA X,SHI Q,SONG X,et al.Tetrachlorobenzoquinone Stimulates NLRP3Inflammasome-Mediated Post-Translational Activation and Secretion of IL-1beta in the HUVEC Endothelial Cell Line[J].Chem Res Toxicol,2016,29(3):421-429)，因此设法抑制内皮细胞IL1β的表达对于抑制内皮细胞炎症反应具有重要的意义。
本公开研究发现干扰内源性TM9SF1基因后，明显抑制HUVEC IL1β表达水平，提示TM9SF1在内皮细胞炎症过程中可能发挥了正向调节作用。
因此，TM9SF1基因可以作为靶点在筛选用于治疗或抑制血管内皮细胞炎症的药物中进行应用。所筛选出的药物通过以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达，间接地实现对IL1β基因表达水平的抑制，起到治疗或抑制血管内皮细胞炎症的作用。
进一步地，上述应用包括：
将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的IL1β表达水平，其中，在存在所述候选试剂情况下，所述IL1β表达水平低于不存在 所述候选试剂情况下的IL1β表达水平，是所述候选试剂作为治疗或抑制血管内皮细胞炎症的药物的指示。
其中，候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平。
进一步地，在本公开的一些实施方案中，该候选试剂可以是针对TM9SF1基因的siRNA；也可以是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
进一步地，在本公开的一些实施方案中，上述生物样品可以是人脐静脉内皮细胞，也可以是鼠脐静脉内皮细胞。
第二方面，本公开提供了抑制或沉默TM9SF1基因表达的试剂在制备用于治疗或抑制血管内皮细胞炎症的药物中的应用。
基于上述发现，抑制或沉默TM9SF1基因表达的试剂可以在制备用于治疗或抑制血管内皮细胞炎症的药物中进行应用，作为一种新的用途。为治疗或抑制血管内皮细胞炎症提供一种新的思路和手段。
进一步地，上述试剂是TM9SF1基因的siRNA。
进一步地，上述siRNA的碱基序列如下所示：
5’-GGUUACGACCUGACGAGUU TT-3’(SEQ ID NO.1)。
其3’端的两个“TT”碱基(下划线)用于提高siRNA稳定性，第1-19位用于与靶基因作用。
具有SEQ ID NO.1所示序列的siRNA能够有效地抑制TM9SF1基因表达，转染该siRNA的细胞，TM9SF1基因的相对表达量为(0.11±0.04)，P<0.005)，远低于对照组，其干扰效率大于50％。与此同时，IL1β基因的表达量为(0.30±0.09，(P<0.001))，明显低于对照组。说明该siRNA的具有较高的干扰效率，且还可作为一种全新的用于治疗或抑制血管内皮细胞炎症的药物。
第三方面，本公开提供了TM9SF1基因作为靶点在筛选用于抑制肿瘤组织血管生成的药物中的应用。
进一步地，上述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
IL8的重要来源细胞之一是内皮细胞，其本身也是内皮细胞炎症的重要参与者之一(BORGES L E,BLOISE E,DELA C C,et al.Urocortin 1expression and secretion by human umbilical vein endothelial cells:In vitro effects of interleukin 8,interferon gamma,lipopolysaccharide,endothelin 1,prostaglandin F-2alpha,estradiol,progesterone and dexamethasone[J].Peptides,2015,74:64-69.)，对内皮细胞迁移(JU L,ZHOU Z,JIANG B,et al. Autocrine VEGF and IL-8Promote Migration via Src/Vav2/Rac1/PAK1Signaling in Human Umbilical Vein Endothelial Cells[J].Cell Physiol Biochem,2017,41(4):1346-1359.)和肿瘤组织血管生成具有促进作用，抑制IL8的表达可以抑制肿瘤组织血管生成(AALINKEEL R,NAIR B,CHEN C K,et al.Nanotherapy silencing the interleukin-8gene produces regression of prostate cancer by inhibition of angiogenesis[J].Immunology,2016,148(4):387-406.MATSUO Y,OCHI N,SAWAI H,et al.CXCL8/IL-8and CXCL12/SDF-1alpha co-operatively promote invasiveness and angiogenesis in pancreatic cancer[J].Int J Cancer,2009,124(4):853-861.)。
本公开研究发现干扰内源性TM9SF1后HUVEC IL8表达水平明显下降，从反面说明TM9SF1可以促进HUVEC细胞IL8的表达。因此，TM9SF1基因可以作为靶点在筛选用于抑制肿瘤组织血管生成的药物中进行应用。所筛选出的药物通过以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达，间接地实现对IL8基因表达水平的抑制，起到抑制肿瘤组织血管生成的作用。
进一步地，该应用包括：
将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的IL8表达水平，其中，在存在所述候选试剂情况下，所述IL8表达水平低于不存在所述候选试剂情况下的IL8表达水平，是所述候选试剂作为抑制肿瘤组织血管生成的药物的指示。
其中，候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平。
TM9SF1基因的cDNA序列如SEQ ID NO.2所示。
进一步地，在本公开的一些实施方案中，该候选试剂可以是针对TM9SF1基因的siRNA；也可以是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
进一步地，在本公开的一些实施方案中，上述生物样品可以是人脐静脉内皮细胞，也可以是鼠脐静脉内皮细胞。
第四方面，本公开提供了抑制或沉默TM9SF1基因表达的试剂在制备用于抑制肿瘤组织血管生成的药物中的应用。
基于上述发现，抑制或沉默TM9SF1基因表达的试剂可以在制备用于抑制肿瘤组织血管生成的药物中进行应用。将抑制或沉默TM9SF1基因表达的试剂作为一种新的用途，为抑制肿瘤组织血管生成提供一种新的思路和手段。
进一步地，上述试剂是TM9SF1基因的siRNA。
进一步地，上述siRNA的碱基序列如下所示：
5’-GGUUACGACCUGACGAGUUTT-3’(SEQ ID NO.1)。
具有SEQ ID NO.1所示序列的siRNA能够有效地抑制TM9SF1基因表达，转染该siRNA的细胞，TM9SF1基因的相对表达量为(0.11±0.04)，P<0.005)，远低于对照组，其干扰效率大于50％。与此同时，IL8基因的表达量为((0.23±0.17，(P<0.005))，明显低于对照组。说明该siRNA的具有较高的干扰效率，且还可作为一种全新的用于抑制肿瘤组织血管生成的药物。
第五方面，本公开提供了TM9SF1基因作为靶点在筛选用于治疗高血压的药物中的应用。
进一步地，上述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
ACE1又称为CD143，是导致血管收缩、血压升高的重要分子，因此临床上将血管紧张素转化酶抑制剂(ACEI)作为治疗高血压的一线药物(CHIEN S C,OU S M,SHIH C J,et al.Comparative Effectiveness of Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers in Terms of Major Cardiovascular Disease Outcomes in Elderly Patients:A Nationwide Population-Based Cohort Study[J].Medicine(Baltimore),2015,94(43):e1751.KANDA D,TAKUMI T,MIYATA M,et al.Angiotensin-Converting Enzyme Inhibitor Prevents the Worsening of Renal Function in the Late Phase after Percutaneous Coronary Intervention[J].J Atheroscler Thromb,2016,23(2):233-240.SHIH C J,CHEN H T,CHAO P W,et al.Angiotensin-converting enzyme inhibitors,angiotensin II receptor blockers and the risk of major adverse cardiac events in patients with diabetes and prior stroke:a nationwide study[J].J Hypertens,2016,34(3):567-574,575.)。本研究结果提示，干扰内源性TM9SF1后HUVEC ACE1的表达量下降了90％以上，说明TM9SF1对内皮细胞ACE1的表达具有重要的促进作用。
因此，TM9SF1基因可以作为靶点在筛选用于治疗高血压的药物中进行应用。所筛选出的药物通过以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达，间接地实现对ACE1基因表达水平的抑制，起到治疗高血压即降血压的作用。
进一步地，该应用包括：
将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的ACE1表达水平，其中，在存在所述候选试剂情况下，所述ACE1表达水平低于不存 在所述候选试剂情况下的ACE1表达水平，是所述候选试剂作为降血压的药物的指示。
其中，候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平。
进一步地，在本公开的一些实施方案中，该候选试剂可以是针对TM9SF1基因的siRNA；也可以是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
进一步地，在本公开的一些实施方案中，上述生物样品可以是人脐静脉内皮细胞，也可以是鼠脐静脉内皮细胞。
第六方面，本公开提供了抑制或沉默TM9SF1基因表达的试剂在制备用于治疗高血压的药物中的应用。
基于上述发现，抑制或沉默TM9SF1基因表达的试剂可以在制备用于治疗高血压的药物中进行应用。将抑制或沉默TM9SF1基因表达的试剂作为一种新的用途，为治疗高血压提供一种新的思路和手段。
进一步地，所述试剂是TM9SF1基因的siRNA。
进一步地，上述siRNA的碱基序列如下所示：
5’-GGUUACGACCUGACGAGUUTT-3’(SEQ ID NO.1)。
具有SEQ ID NO.1所示序列的siRNA能够有效地抑制TM9SF1基因表达，转染该siRNA的细胞，TM9SF1基因的相对表达量为(0.11±0.04)，P<0.005)，远低于对照组，其干扰效率大于50％。与此同时，ACE1基因的表达量为(0.07±0.01，(P<0.001))，明显低于对照组。说明该siRNA的具有较高的干扰效率，且还可作为一种全新的用于降血压的药物。
基于上述几方面的内容，在第六方面，本公开还提供了一种药物，所述药物具有以下用途中一种或几种：
(1)用于治疗或抑制血管内皮细胞炎症；
(2)用于抑制肿瘤组织血管生成；
(3)用于治疗高血压；
所述药物含有抑制或沉默TM9SF1基因表达的试剂以及药学上可接受的辅料。
进一步地，在本公开的一些实施方案中，上述试剂是针对TM9SF1基因的siRNA；或者，上述试剂是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
进一步地，上述siRNA的碱基序列如下所示：
5’-GGUUACGACCUGACGAGUUTT-3’(SEQ ID NO.1)。
基于上述几方面的内容，在第七方面，本公开还提供了一种方法，该方法为治疗或抑制血管内皮细胞炎症的方法，抑制肿瘤组织血管生成的方法，或者是治疗高血压的方法，其包括：给予主体施用上述第六方面提供的药物。
进一步地，在本公开的一些实施方案中，上述主体为小鼠、猴、或人。
综上，本公开提供的TM9SF1基因作为靶点以及抑制TM9SF1基因的试剂在血管性疾病方面的新用途，为治疗和预防血管性疾病提供了一种新的思路和手段。
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
1材料与方法
1.1细胞及主要试剂
HUVEC购自中科院上海细胞库。TM9SF1特异性siRNA(SEQ ID NO.1)由吉玛公司设计并合成。内皮细胞专用培养基EGM由瑞士LONZA公司生产；胰酶(含EDTA)、胎牛血清(FBS)、磷酸盐缓冲液(PBS)等由美国Hyclone公司生产；转染试剂Lipofectamine 3000购自美国Thermo公司；CD31抗体购买自美国Immunoway公司；免疫细胞化学显色试剂盒购买自北京中杉金桥公司；CYBR Green Mix购自北京康为世纪生物科技有限公司；CCK8购自翊圣公司。
1.2HUVEC细胞培养与鉴定
HUVEC培养于内皮细胞专用培养基EGM中，在37.5℃、5％CO 2、饱和湿度的培养箱中培养，隔天换液，细胞汇合度达到80％时进行传代，用0.25％胰酶消化，待细胞变圆且部分细胞脱离培养皿时用FBS终止消化，吹打混匀，1 200r/min离心5min，沉淀用培养基重悬，计数，接种至新的培养皿。采用免疫细胞化学方法对细胞进行鉴定，HUVEC细胞爬片汇合度至80％时，用4％多聚甲醛固定30min，PBS洗涤后用0.1％TritonX-100打孔30min。山羊血清封闭30min后，兔抗CD31(Immunoway公司，YT0752，1:200稀释)4℃孵育过夜。PBS洗涤后二抗工作液37℃孵育30min，PBS充分洗涤，DAB显色1min，苏木素复染1min，自来水返蓝，中性树胶封片。正置显微镜下观察拍照，CD31主要定位于细胞膜表面，表达阳性细胞呈棕褐色染色。
1.3HUVEC分组及TM9SF1基因干扰
细胞接种至6孔板(5×10 5个细胞/孔)，待第2天细胞贴壁后分为阴性对照组和干扰组进行转染。Lipofectamin 3000转染过程如下：2.5μL Lipofectamin 3000加入50μL PBS中稀释混匀，2.5μL siRNA(SEQ ID NO.1)(20μM)加入50μL PBS 中稀释混匀，两种稀释液轻轻混合，室温放置5min，然后将混合液轻轻滴入培养基，混匀。转染后4～6h换液。
1.4实时荧光定量PCR(qPCR)
细胞转染TM9SF1特异性siRNA(SEQ ID NO.1)后，于48h用Trizol裂解细胞，提取总RNA，取1-3μg逆转录成cDNA，以此为模板，用SBRY Green染料法实时定量PCR检测相关基因的表达水平。qPCR扩增条件：95℃10min，95℃15s，60℃1min，共40个循环。以GAPDH作为内参。基因上下游引物序列分别如下：
Figure PCTCN2018095821-appb-000001
1.5统计学方法
应用GraphPad Prism 5软件分析处理数据，计量资料以均数±标准差表示，两组间比较采用非配对t检验，以P<0.05为差异有统计学意义。
2结果
2.1脐静脉内皮细胞鉴定
相差显微镜下观察HUVEC生长状态良好，如图1所示(图中：A为相差显微镜下观察HUVEC生长情况；B为免疫细胞化学检测CD31表达情况，箭头所示为典型表达强阳性细胞)，细胞呈多边形，有的有少量突起(图1-A)。由于CD31为内皮细胞标志性分子，主要表达于细胞膜，应用免疫细胞化学方法检测了HUVEC细胞CD31的表达情况，结果显示几乎所有细胞均为棕褐色，只是染色深浅不同，提示绝大部分细胞CD31表达阳性，为典型内皮细胞(图1-B)。
2.2 TM9SF1特异性siRNA干扰效果验证
利用qPCR技术检测转染TM9SF1特异性siRNA(SEQ ID NO.1)后TM9SF1基因的相对表达量，结果如图2所示(图中：si-NC为阴性对照组，si-TM9SF1为转染siRNA的干扰组，**表示P<0.005)。转染48h后，以阴性对照组(si-NC)为参照标准1，si-TM9SF1组TM9SF1基因的相对表达量为(0.11±0.04)，P<0.005)，干扰效率大于50％，说明该siRNA有效。
2.3干扰TM9SF1抑制HUVEC炎症相关基因的表达
结果如图3所示(图中：A为IL1β基因相对表达量；B为IL8基因相对表达量；C为ACE1基因相对表达量；**P<0.005或***P<0.001)，以阴性对照组为参照标准1，干扰组IL1β、IL8和ACE1相对表达量分别为(0.30±0.09，(P<0.001))，(0.23±0.17，(P<0.005))和(0.07±0.01，(P<0.001))，表达水平受到明显抑制。
本公开利用TM9SF1特异性siRNA干扰该基因内源性表达，通过实时定量PCR技术对其干扰效果进行了验证，并发现与内皮细胞功能密切相关的基因IL1、IL8和ACE1表达明显下降(P<0.005)，上述结果提示TM9SF1基因可能对内皮细胞功能具有重要调节作用。
这就说明，TM9SF1基因可以作为一种新的靶点，可将其应用于筛选用于治疗或抑制血管内皮细胞炎症(与IL1β基因表达相关)的药物、用于抑制肿瘤组织血管生成(与IL8基因表达相关)的药物、用于治疗高血压(与ACE1基因表达相关)的药物等领域中；
此外，抑制或沉默TM9SF1基因表达的试剂(例如具有SEQ ID NO.1所示的siRNA)可用于制备用于治疗或抑制血管内皮细胞炎症的药物、用于抑制肿瘤组织血管生成的药物以及用于治疗高血压的药物等领域中。
总之，TM9SF1基因可以作为靶点在血管性疾病方面的新用途，为治疗和预防血管性疾病提供了一种新的思路和手段。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性：
本公开的研究揭示了通过抑制或沉默TM9SF1基因的表达，可抑制或沉默IL1β、IL8以及ACE1基因的表达，因此，利用这一发现，可以将TM9SF1基因作为靶点，用于到筛选治疗或抑制血管内皮细胞炎症的药物、筛选用于抑制肿瘤组织血管生成和筛选用于治疗高血压的药物等领域中，同时，抑制或沉默TM9SF1基因表达的试剂也可以用于制备治疗 或抑制血管内皮细胞炎症的药物、抑制肿瘤组织血管生成的药物和治疗高血压的药物等领域中。

Claims (20)

  1. TM9SF1基因作为靶点在筛选用于治疗或抑制血管内皮细胞炎症的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
  3. 根据权利要求1或2所述的应用，其特征在于，所述应用包括：
    将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
    将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
    确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的IL1β表达水平，其中，在存在所述候选试剂情况下，所述IL1β表达水平低于不存在所述候选试剂情况下的IL1β表达水平，是所述候选试剂作为治疗或抑制血管内皮细胞炎症的药物的指示。
  4. 根据权利要求3所述的应用，其特征在于，所述候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平。
  5. 根据权利要求4所述的应用，其特征在于，所述候选试剂是针对TM9SF1基因的siRNA；
    或者，所述候选试剂是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
  6. 抑制或沉默TM9SF1基因表达的试剂在制备用于治疗或抑制血管内皮细胞炎症的药物中的应用。
  7. TM9SF1基因作为靶点在筛选用于抑制肿瘤组织血管生成的药物中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
  9. 根据权利要求7或8所述的应用，其特征在于，所述应用包括：
    将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
    将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
    确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的IL8表达水平，其中，在存在所述候选试剂情况下，所述IL8表达水平低于不存在所述候选试剂情况下的IL8表达水平，是所述候选试剂作为抑制肿瘤组织血管生成的药物的指示。
  10. 根据权利要求9所述的应用，其特征在于，所述候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平。
  11. 根据权利要求10所述的应用，其特征在于，所述候选试剂是针对TM9SF1基因的siRNA；
    或者，所述候选试剂是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
  12. 抑制或沉默TM9SF1基因表达的试剂在制备用于抑制肿瘤组织血管生成的药物中的应用。
  13. TM9SF1基因作为靶点在筛选用于治疗高血压的药物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述药物以TM9SF1基因为靶点，抑制或者沉默TM9SF1基因的表达。
  15. 根据权利要求13或14所述的应用，其特征在于，其包括：
    将含有TM9SF1基因的生物样品在存在候选试剂的情况下培养；
    将含有TM9SF1基因的生物样品在不存在所述候选试剂的情况下培养；
    确定上述生物样品在存在所述候选试剂和不存在所述候选试剂的情况下的ACE1表达水平，其中，在存在所述候选试剂情况下，所述ACE1表达水平低于不存在所述候选试剂情况下的ACE1表达水平，是所述候选试剂作为降血压的药物的指示。
  16. 根据权利要求15所述的应用，其特征在于，所述候选试剂是以TM9SF1基因为靶点，抑制或沉默其表达水平；
    优选的，所述候选试剂是针对TM9SF1基因的siRNA；或者，所述候选试剂是针对TM9SF1蛋白的抗体，其可在蛋白水平上抑制TM9SF1蛋白的活性或数量。
  17. 抑制或沉默TM9SF1基因表达的试剂在制备用于治疗高血压的药物中的应用。
  18. 根据权利要求6、12或17所述的应用，其特征在于，所述试剂是TM9SF1基因的siRNA。
  19. 一种药物，所述药物具有以下用途中一种或几种：
    (1)用于治疗或抑制血管内皮细胞炎症；
    (2)用于抑制肿瘤组织血管生成；
    (3)用于治疗高血压；
    其特征在于，所述药物含有抑制或沉默TM9SF1基因表达的试剂以及药学上可接受的辅料。
  20. 一种方法，该方法是治疗或抑制血管内皮细胞炎症的方法、抑制肿瘤组织血管生成的方法、或者是治疗高血压的方法，其特征在于，其包括：给予主体施用权利要求19所述的药物。
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