WO2017197972A1 - 氯福克酚用于制备治疗人神经胶质瘤的药物的用途 - Google Patents

氯福克酚用于制备治疗人神经胶质瘤的药物的用途 Download PDF

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WO2017197972A1
WO2017197972A1 PCT/CN2017/077346 CN2017077346W WO2017197972A1 WO 2017197972 A1 WO2017197972 A1 WO 2017197972A1 CN 2017077346 W CN2017077346 W CN 2017077346W WO 2017197972 A1 WO2017197972 A1 WO 2017197972A1
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glioma
cells
cell
clofoctol
human
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French (fr)
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彭小忠
胡艳
韩为
强伯勤
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中国医学科学院基础医学研究所
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Priority to EP17798537.1A priority Critical patent/EP3459537B1/en
Priority to US16/303,138 priority patent/US10987318B2/en
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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A61K31/055—Phenols the aromatic ring being substituted by halogen
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the invention relates to the field of tumor treatment, in particular to the use of chlorfluoxol for preparing a medicament for treating human glioma.
  • Glioma is the most common primary intracranial malignant tumor derived from the neuroepithelial. It has high malignancy, rapid growth, strong invasiveness and high morbidity. Although the current comprehensive treatment technology for cancer has made great progress, it is located in an important position in the brain, accompanied by microvascular proliferation, showing obvious invasive growth characteristics. Traditional surgery and radiotherapy cannot be completely cured, resulting in high recurrence rate. High mortality and low cure rate.
  • the drugs for clinical chemotherapy are mainly alkylated drugs, but the traditional alkylating agents have large side effects and are easy to produce drug resistance; the new alkylating agent temozolomide (TMZ) has small side effects and long-term drug tolerance.
  • TMZ new alkylating agent temozolomide
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • Inhibitors such as EGFR gefitinib and erlotinib have been shown to be highly susceptible to drug resistance after administration, and patients with efficacy are very rare, and progression-free survival has not been extended after taking the patient.
  • EGFR/Her2 inhibitor lapatinib, EGFR monoclonal antibody cetuximab has been confirmed to have a relatively small effect on glioma; in addition to mTOR related inhibitors: rapamycin, for Sirolimus, everolimus, dexamethasone, deoxyacetylase (Histone deactylase, HDAC) inhibitors: valerinostat alone or in combination with temozolomide have been shown to have no or minimal effect .
  • the study also developed a number of anti-angiogenic molecules such as bevacizumab and cediranib that have been shown to have partial effects in response to the rich microvascular formation of gliomas, which can rapidly reduce para-carcinoma and effectively It prolongs the patient's progression-free survival, but it does not reverse the progression of the disease.
  • Patients who received anti-vascular therapy eventually failed to cure successfully, nor could they change the status of death in most patients months after diagnosis.
  • no new and effective drug molecules have been found. Due to the complex pathogenesis of glioma, we do not know which key points in glioma patients caused by different pathogenesis.
  • glioma stem cells are the root cause of glioma development, and glioma stem cells can induce tumor angiogenesis by activating Wnt signaling pathway, enhancing tumor hypoxia stress and invasion ability.
  • Wnt signaling pathway enhancing tumor hypoxia stress and invasion ability.
  • Bao et al. confirmed that glioma stem cells can also produce chemoradiation resistance.
  • the chemotherapeutic drugs of glioma only target the whole tumor tissue, and the patient has a certain effect after shrinking the tumor tissue after receiving the treatment for a period of time, but the glioma stem cells resistant to chemotherapy can be further formed by proliferation and differentiation. New tumor tissue that promotes tumor recurrence.
  • Chlorfofol is an antibacterial drug mainly used to treat upper respiratory tract, ear, nose and throat infections caused by Gram-positive bacteria. Currently, it is mainly used in France and Italy, and it was first approved by France in 1980.
  • the inhibition of Gram-positive bacteria may be achieved by inhibiting the synthesis of cell wall and affecting energy metabolism, but it does not affect the synthesis of DNA, RNA and protein.
  • the resistance of Gram-negative bacteria to chlorofosphenol may be due to its inability to pass the outer membrane of Gram-negative bacteria.
  • the administration method is an oral administration of a rectal suppository (1.5 g/person, daily, main administration method).
  • the present inventors have found that four glioma stem cell lines isolated from the glioma cell line, human normal astrocyte cell line, human embryonic kidney cell, human neural stem cell and chlorfluoxol at the cellular level have Specific inhibition effect.
  • the present invention verifies at the cellular level that chlorofosphenol can specifically inhibit the self-renewal ability and tumor ball formation ability of glioma stem cells; and the chlorford is verified by zebrafish and nude mouse glioma transplantation model. The therapeutic effect of gramol on glioma.
  • chlorfluoxol will likely provide a new potent therapeutic for the treatment of gliomas.
  • the present invention provides a use of clofibrate for the preparation of a medicament for treating human glioma.
  • the medicament treats the human glioma by inhibiting the cellular activity of glioma stem cells.
  • the medicament treats the human glioma by inhibiting the ability of glioma stem cells to self-renew, tumor ball formation, and tumor formation in vivo.
  • the drug treats the human glioma by inducing apoptosis of glioma stem cells.
  • the glioma stem cells include, but are not limited to, U87MG SLC, U251SLC, GSC2, GSC5.
  • the medicament further treats the human glioma by inhibiting glioma cells.
  • the glioma cells include, but are not limited to, U251, U87MG, N3.
  • the human glioma is at grades I to IV.
  • Glioma stem cells a very rare class of cells with self-renewal ability, multiple differentiation ability, and tumor formation ability in glioma tissues, which develops, relapses and gels with gliomas. Stromal tumors are closely related to the formation of resistance to chemotherapy and chemotherapy.
  • MTS Method A detection reagent for detecting the number of viable cells in cell proliferation and cytotoxicity experiments by colorimetry.
  • MTS is a novel tetrazole compound and PMS is an electronic coupling agent. PMS has enhanced chemical stability which allows it to be mixed with MTS to form a stable solution.
  • MTS is biologically reduced to a colored formazan product that is directly soluble in the medium. This transformation is most likely accomplished by the action of NADPH or NADH produced by dehydrogenase in metabolically active cells. The amount of formazan product detected at 490 nm is directly proportional to the number of viable cells in the culture.
  • Self-renewal refers to stem cells (here specifically referred to as glioma stem cells) through symmetric or asymmetric division Producing at least one process of retaining stem cell trait daughter cells, self-renewal can maintain the multipotential potential of stem cells. For tissue-specific stem cells, self-renewal is the basis for maintaining their potential for differentiation throughout life.
  • Tumor sphere forming ability refers to the ability of tumor stem cells (here specifically referred to as glioma stem cells) to be cultured in a medium containing EGF and bFGF in vitro to form a tumor sphere.
  • the present invention finds that the clinical drug clofibrate can be used for the preparation of a medicament for treating human glioma, thereby providing a new powerful drug for the treatment of human glioma, with significant social and economic benefits.
  • FIG. 1 Chlorofolol specifically inhibits the cellular activity of glioma stem cells.
  • MTS tetrazolium
  • glioma stem cell lines U87MG SLC, U251SLC, GSC2, GSC5
  • three gums A tumor cell line (U251, U87MG, N3 (primary glioma cells isolated from patient tumor tissue)
  • three human normal cell lines human normal astrocytes (HA), human neural stem cells (H1p56NSC)
  • H1p56NSC human embryonic kidney cells
  • Chlorfokolol specifically inhibits glioma stem cells.
  • the green fluorescently labeled GSC2-GFP (Fig. 2A) or U87MG SLC-GFP (Fig. 2B) was mixed with the non-fluorescently labeled HA 1:1 in the system, and the chlorofosphenol was at 1 ⁇ M and 3 ⁇ M, respectively.
  • the flow cytometry was used to analyze the ratio of GSC2-GFP or U87MG SLC-GFP and HA in the viable cells of different treatment groups, and combined with the cells in different concentrations and different time in the co-culture model. Survival rate, statistical analysis of the relative cell viability of each cell component in the co-culture model.
  • FIG. 3A to Figure 3H Chlorfokolol inhibits the growth and maintenance of glioma stem cell tumor spheres.
  • 100 ⁇ L of single cell suspension containing 5000 GSC2 was added to each well of a 96-well cell culture dish. After 4 days of natural growth, the original medium was aspirated, and 100 ⁇ L of fresh medium containing each concentration of the drug was added to each well, and the drug concentration was respectively determined.
  • 0 ⁇ M control, Figure 3A
  • Figure 3B 0.03 ⁇ M
  • Figure 3C Figure 3C
  • 0.3 ⁇ M Figure 3D
  • 1 ⁇ M Figure 3E
  • 3 ⁇ M Figure 3F
  • 10 ⁇ M Figure 3G
  • 30 ⁇ M Fig.
  • FIG. 5 Chlorfokolol inhibits the ability of glioma stem cells to self-renew.
  • the GSC5 cells were diluted to 25, 50, 100, 200/100 ⁇ L, and chlorfluoxol was treated at a concentration of 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M for 2 weeks, and then the percentage of tumor-free spherical cells was counted, and the ordinate indicates the percentage of tumor-free spherical cells. The logarithmic value.
  • Figures 6A and 6B The effect of chlorofosphenol on glioma stem cells is somewhat irreversible.
  • Drug treatment GSC2 24 hours, 48 hours after the withdrawal of the drug, replaced with fresh medium to continue to cultivate 48 small
  • the survival rate of each treatment group was measured by the method of MTS, and the changes in cell viability of the treatment group were compared with those of the drug treatment group.
  • “1 day” and “2 days” means MTS detection after 24 hours or 48 hours of drug treatment; "drug continued action” means withdrawal of the drug after 24 hours and 48 hours of drug treatment, and continued to add fresh medium culture with the corresponding drug concentration 48 After the hour, the MTS test; “withdrawal of the drug” means that the drug was withdrawn after 24 hours and 48 hours of drug treatment, and the fresh medium containing no drug was added to continue the culture for 48 hours after the MTS test.
  • Fig. 7A and Fig. 7B clofibrate can significantly inhibit the in situ tumor sphere formation ability of nude mice at a concentration of 30 ⁇ M.
  • the chlorofosphenol was pretreated with GSC2 at a concentration of 30 ⁇ M for 24 hours, then the viable cells were collected, and some cells were counted after trypan blue staining, and the cells were intracranially tumorigenic, 10 5 viable cells/only (5 nude mice) ), and set the DMSO control group (5 nude mice), the DMSO treatment group (Fig. 7A), and the chlorfluoxol pretreatment group (Fig. 7B) to observe the results of HE staining in situ in nude mice.
  • FIGs 8A and 8B Chlorfokolol significantly inhibited tumor growth in a zebrafish glioma xenograft model.
  • the yolk sac of the 48-hour zebrafish embryo was injected with 3000 green fluorescently labeled U87MGSLC-GFP, which was naturally grown for 48 hours, and treated with chlorfluoxol at a concentration of 10 ⁇ M for 48 hours (Fig. 8B). Fluorescence microscopy was used to detect zebrafish. The intensity of green fluorescence in the yolk sac area was used to determine the anti-tumor effect of clofibrate on the zebrafish xenograft model.
  • Figure 8A is the control group.
  • chlorofosphenol can significantly inhibit tumor growth in a plaque nude mouse xenograft model with less side effects.
  • GSC2 cells were inoculated into 15 subcutaneous subcutaneouss of 5 weeks old Balb/C nude mice, and the number of inoculations was 10 5 per mouse.
  • the phenolic drug-administered group was administered at a dose of 20 mg/kg, administered intraperitoneally for 11 days, during which time the volume of tumors (Fig. 9A and Fig. 9B) and body weight (Fig. 9C) of the two nude mice were recorded. .
  • FIG. 10A-1 to Figure 10A-5 Chlorfokolol induces apoptosis in glioma stem cells. Chlorofosphenol treatment of GSC2 at 0 ⁇ M (control, Figure 10A-1), 1 ⁇ M (Figure 10A-2), 3 ⁇ M ( Figure 10A-3), 10 ⁇ M ( Figure 10A-4), 30 ⁇ M ( Figure 10A-5) After 12 hours, Annexin V/PI double staining, flow analysis of cell apoptosis in each treatment group, Figure 10A-1 to Figure 10A-5 (Gate: P2) found that chlorfluoxol can significantly induce glioma stem cells Apoptosis.
  • FIG. 10B-1 to Figure 10B-5 Chlorofol can induce apoptosis of glioma stem cells. Chlorofosphenol was treated at a concentration of 0 ⁇ M (control, Figure 10A-1), 1 ⁇ M (Figure 10A-2), 3 ⁇ M ( Figure 10A-3), 10 ⁇ M ( Figure 10A-4), 30 ⁇ M ( Figure 10A-5). After an hour, Annexin V/PI double staining, flow analysis of cell apoptosis in each treatment group, Figure 10B-1 to Figure 10B-5 (Gate: P2) found that chlorfluoxol can significantly induce glioma stem cells Apoptosis.
  • a, DPBS sequentially add 0.2g KCl, 8.0g NaCl, 0.2g KH 2 PO 4 , 1.15g Na 2 HPO 4 to 1L, adjust the pH to 7.35, then add 0.1gMgCl 2 .6H 2 O, mix well until the solution is clear, then add after 0.133gCaCl 2 .2H 2 O, this solution was thoroughly mixed until clear. After sterilizing twice with a 0.2 ⁇ M filter, the mixture was stored at 4 ° C;
  • PMS solution Prepare a 0.92 mg/mL PMS solution using DPBS, filter and sterilize twice with a 0.2 ⁇ M filter, and dispense in an EP tube wrapped with tin-plated paper, and store at -20 ° C in the dark;
  • MTS solution Prepare according to the ratio of 1 mg MTS powder dissolved in 0.5 mL DPBS, mix gently, about 15 min, until MTS is completely dissolved. The pH was measured and stored between 6.0 and 6.5. After sterilizing twice with a 0.2 ⁇ M filter, the mixture was placed in an EP tube wrapped with tin-plated paper, and stored at -20 ° C in the dark;
  • MTS/PMS solution MTS and PMS were separately thawed, incubated at 37 ° C for 15 min, then dissolved in 2.1 mL of DPBS according to 4.2 mg of MTS powder, and 0.1 mL of PMS was added at the time of use.
  • the chemical formula of chlorofosphenol was C 21 H 26 Cl 2 O, and the structural formula was Clc1cc(Cl)ccc1Cc2cc(ccc2O).
  • concentration gradients were set: 0.3 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M, and 3 replicate wells per drug concentration for 48 hours.
  • glioma cell line (U251 (purchased from ATCC), U87MG (purchased from ATCC, USA), N3 (primary glioma cells isolated from patient tumor tissue, received from Tiantan Hospital, Jiang Tao) Teacher's laboratory)), three normal human cell lines (human normal astrocytes (HA, purchased from ATCC), human neural stem cells (H1p56NSC, cell strain established in our laboratory), human embryonic kidney cells (293ET) It was purchased from the Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences.) It was not coated with 96-well cells for coating. The cell plating concentration was 5000/well. Other operations are as above.
  • chlorfluoxol specifically inhibits the cell activity of glioma stem cells.
  • Example 2 Eukaryotic cell lentivirus infection
  • the U87MG SLC cells stably expressing green fluorescence were screened with the corresponding concentration of blasticidin, and the GSC2 cells stably expressing green fluorescence were screened with the corresponding concentration of puromycin, and the method of selecting the monoclonal was selected.
  • HA cells stably expressing red fluorescence were screened.
  • 6-well and 96-well cell culture plates were pre-coated with 100 ⁇ g/mL polylysine, washed overnight at 37 ° C, washed twice with physiological saline, and air-dried.
  • GSC2-GFP cells stably expressing green fluorescence and HA cells stably expressing red fluorescence were digested into single cells, resuspended in fresh medium, and counted in cells, diluted to 5 ⁇ 10 4 cells/mL, and the two cells were 1:1.
  • Mix well divide into five tubes, add chlorfluoxol, and mix them to a concentration of 0 ⁇ M, 1 ⁇ M, 3 ⁇ M, 10 ⁇ M, 30 ⁇ M.
  • 6-well cell culture plates (2 mL) and 96-well cell culture plates (100 ⁇ L, 3 replicate wells per group) were added to each other for 24 hours, and the cell viability of each treatment group in 96-well cell culture plates was determined by MTS method.
  • the cells in the 6-well cell culture plates were separately collected, washed twice with PBS, and resuspended with 100 ⁇ L each, and the proportion of GSC2-GFP and HA-RFP in the living cells of each treatment group was analyzed by flow cytometry.
  • the relative cell viability of each cell component in the co-culture model was statistically analyzed by combining the co-culture model with the changes in cell viability after drug treatment.
  • Co-culture model - U87MG SLC-GFP + HA-RFP operates as above;
  • Chlorfluoxol specifically inhibits the cell viability of glioma stem cells in a co-culture model relative to human normal astrocytes.
  • Fig. 3A to Fig. 3H and Fig. 4 The results are shown in Fig. 3A to Fig. 3H and Fig. 4, and the in vitro experiment was used to simulate the inhibitory effect of clofibrate on the formation of tumors in clinical glioma stem cells. It was found that chlorfluoxol can significantly reduce the volume and number of tumor spheres. And low concentration (0.03 ⁇ M, 0.1 ⁇ M) GSC2 can grow protrusions, showing obvious differentiation-like characteristics. At high concentration (10 ⁇ M, 30 ⁇ M), the tumor ball edge cells can die and reach the entire tumor sphere.
  • the suspension cultured GSC5 tumor spheroid cells were collected into a 15 mL centrifuge tube, and 0.5 mLACCUTASE enzyme (purchased from sigma) was added for digestion at 37 ° C for 5 mins, the cells were blown into single cells, and the supernatant was centrifuged;
  • the cells were seeded in a 96-well plate, 100 ⁇ L/well, 37 ° C, 5% CO 2 , cultured in an incubator, and the ratio of tumor-free spherical wells was counted after 2 weeks;
  • the drug was allowed to act for 48 hours, and then the test was resumed for 48 hours, as described above.
  • the cells were pretreated for 24 hours after clofibrate at 30 ⁇ M, washed twice with PBS, partially stained with trypan blue, and counted.
  • the cells were injected in situ into the intracranial striatum of Balb/C nude mice at 5-7 weeks old, 10 5 cells/only, 5 rats in each group, and a DMSO control group was set up.
  • Example 8 Administration of zebrafish glioma transplantation model
  • 3000 green fluorescein-labeled U87MGSLC-GFP was injected into the yolk sac of 48-hour zebrafish embryos, allowing them to grow naturally for 48 hours. Fluorescence microscopy was used to detect tumor formation in zebrafish, and then chlorine was added to the zebrafish hatching solution. The gramol was measured at a concentration of 10 ⁇ M for 48 hours. The fluorescence intensity of the green fluorescence of the zebrafish yolk sac was detected by fluorescence microscopy to determine the inhibitory effect of clofibrate on the tumor in the zebrafish xenograft model.
  • chlorfluoxol significantly inhibited tumor growth in a nude mouse xenograft model, and its side effects were small.
  • Annexin V/PI double staining kit used was purchased from BD pharmingen with product lot number 556547.
  • the present invention demonstrates chlorfromycin at the cellular level relative to glioma cells (U251, U87MG, N3 (primary glioma cells isolated from patient tumor tissue)), human normal astrocytes (HA), human embryonic kidney cells (293ET), human neural stem cells (H1P56NSC) have significant specific inhibitory effects on four glioma stem cells (U87MG SLC, U251SLC, GSC2, GSC5) isolated from the laboratory (48 hours effect) The IC 50 value is: 7.65 to 10.53 ⁇ M).
  • the present invention utilizes a zebrafish glioma xenograft model and a nude mouse glioma subcutaneous xenograft model to demonstrate the therapeutic effect of clofibrate on glioma at the body level.
  • chlorfluoxol significantly induces apoptosis of glioma stem cells, and explains to some extent the inhibitory effect of clofibrate on glioma stem cells and its anti-tumor effect on glioma.
  • the present invention finds for the first time that the clinical drug clofibrate can achieve the purpose of treating human glioma by inhibiting human glioma stem cells. It is well known that small molecule compounds rely on related key groups to function in the body, and on the basis of this, other group changes can produce different compounds, but they can also perform their intended functions. Therefore, any modification to chlorofosphenol based on this, a modification to the same effect as the present invention is used in the present invention.

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Abstract

本发明公开了氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中氯福克酚是一种治疗上呼吸道感染药物，其相对于人神经胶质瘤细胞及人正常星形胶质细胞、人胚肾细胞，人神经干细胞，可显著特异性抑制胶质瘤干细胞活性，且经氯福克酚处理后胶质瘤干细胞的自我更新、肿瘤球形成、裸鼠体内成瘤能力也明显降低。体内实验，利用斑马鱼胶质瘤移植瘤模型、裸鼠胶质瘤移植瘤模型，在体验证该药物对胶质瘤的治疗效果。具体而言，本发明发现用于治疗上呼吸道感染药物——氯福克酚可通过靶向性抑制胶质瘤干细胞达到用于制备治疗人神经胶质瘤的药物目的。本发明为人神经胶质瘤的治疗提供了新的治疗药物。

Description

氯福克酚用于制备治疗人神经胶质瘤的药物的用途 技术领域
本发明涉及肿瘤治疗领域，具体涉及氯福克酚用于制备治疗人神经胶质瘤的药物的用途。
背景技术
神经胶质瘤是最常见的来源于神经上皮的原发性颅内恶性肿瘤，其恶性程度高、生长迅速、浸润性强，具有高发病率的特点。虽然目前肿瘤综合治疗技术已取得长足进步，但因其位于脑部重要位置，且伴随微血管增生，呈现明显的浸润性生长等特点，传统的手术、放疗都无法彻底根治，而导致其复发率高、病死率高和治愈率低。目前临床上化疗的药物以烷基化药物为主，但传统烷化剂副作用大，易产生耐药性；新型烷化剂替莫唑胺(temozolomide，TMZ)虽毒副作用小，且长时间用药耐受性好，但TMZ治疗后引起的骨髓增生异常综合征(myelodysplastic syndrome，MDS)、急性髓性白血病(acute myeloid leukemia，AML)以及急性成淋巴细胞性白血病(acute lymphoblastic leukemia，ALL)的报道与日俱增，具体原因也在进一步研究中。因此，寻找新一代治疗神经胶质瘤的有效药物，改善神经胶质瘤的治疗迫在眉睫。
近年来，科学家们结合胶质瘤的发生发展的机理，在其新药探索与开发方面做出了诸多努力。研究显示，神经胶质瘤的发生发展主要与RTK/RAS/PI3K通路，p53通路，RB通路中的相应关键分子的异常表达或突变相关。因而近年来的研究都在开发相应的药物分子的抑制剂用以抵消某种关键分子的作用效果，但都只是在临床前研究结果中表现出强的治疗前景，在Ⅰ、Ⅱ期临床结果，或是批准上市后表现得不尽人意或疗效颇微。如EGFR的抑制剂：吉非替尼和埃罗替尼被证实患者服用后极易产生耐药，而且有疗效的患者非常罕见，并且病人服用之后无进展生存期皆未有所延长。同时EGFR/Her2抑制剂拉帕替尼，EGFR的单克隆抗体西妥昔单抗都被证实对胶质瘤的治疗效果颇微；除此以外还有mTOR相关抑制剂：雷帕霉素、替西罗莫司、依维莫司、地磷莫司，脱乙酰酶(Histone deactylase，HDAC)抑制剂：伏立诺他等单独治疗或与替莫唑胺联合治疗都被证实没有或只有极小的作用效果。与此同时，研究还针对胶质瘤微血管形成丰富的特点，开发了被证实有部分效果的抗血管生成的分子如贝伐单抗、西地尼布，它们可以快速的减少癌旁水肿，有效的延长了患者的无进展生存期，但是也无法逆转病程的进展。接受抗血管疗法的患者最终也无法成功治愈，还是无法改变大多数患者在诊断后数月死亡的现状。总体而言，尽管国内外科学家，药物公司在胶质瘤的新药探索中付出诸多人力物力，目前都没有找到一种新的，有效的药物分子。由于胶质瘤复杂的发病机制，目前我们尚不知道在不同发病机理导致的胶质瘤患者中哪个关键分 子需要被抑制。此外，另一些科学家提出，胶质瘤组织内细胞存在异质性，以上探索的药物分子之所以结果颇微，可能与我们没有针对对胶质瘤的发生、发展起到重要作用的细胞类型有关。
1994年，Lapidot等从白血病患者身上首次分离获得肿瘤干细胞，肿瘤干细胞理论逐渐被接受。随后Singh等在胶质瘤中分离得到极少数具有自我更新及分化能力的CD133+胶质瘤干细胞，且其相对于CD133-的细胞具有更强的体内成瘤能力，仅100个细胞就可在裸鼠内诱导形成肿瘤。Jian Chen等通过细胞体内示踪技术，从在体的水平，证明了胶质瘤干细胞的存在。随着研究的深入，胶质瘤干细胞的特点性质，以及与胶质瘤的关系逐渐被揭示。研究表明胶质瘤干细胞是胶质瘤发生发展的根本原因，且胶质瘤干细胞可通过激活Wnt信号通路诱导肿瘤血管形成，增强肿瘤的低氧应激以及侵袭能力等。不仅如此，Bao等证实胶质瘤干细胞也能产生放化疗抗性。然而，目前神经胶质瘤的化疗药物只针对整个肿瘤组织，病人在接受一段时间的治疗后，肿瘤组织缩小取得一定的效果，但是对化疗产生抵抗的胶质瘤干细胞可通过增殖和分化进一步形成新的肿瘤组织，从而促使肿瘤复发。由此可见，胶质瘤干细胞与胶质瘤的发生发展，及放化疗抗性的形成，肿瘤的复发息息相关，是胶质瘤难以攻克的一个重要因素。因此，我们设想，是否可以通过获得靶向性胶质瘤干细胞的药物，并结合传统的放化疗，达到最终彻底消灭整个肿瘤组织的目的。
近几年，国内外科学家分别在不同肿瘤中报道了几种能靶向肿瘤干细胞的药物，其中包括一些新的药物分子和已在临床上使用的药物分子。如天然的多酚白藜芦醇，具有抗菌活性的离子载体盐霉素，衣霉素，灭绦虫活性的药物氯硝柳胺，以及用以治疗糖尿病的药物二甲双胍及用以治疗肝癌的药物索拉非尼，都利用裸鼠成瘤模型，验证了药物相对于目前的一线药物TMZ更具抑瘤效果。但是以上药物都无法在保证安全性的前提下，发挥靶向肿瘤干细胞的功能，因而效果有限。
氯福克酚是一种抗菌药物，主要用于治疗有革兰氏阳性菌引起的上呼吸道，耳，鼻，喉的感染。目前，主要用于法国和意大利，其首先法国在1980年期间批准上市。该要对革兰氏阳性菌的抑制可能是通过抑制胞壁质的合成、影响能量代谢实现的，但它不影响DNA、RNA及蛋白的合成。而革兰氏阴性菌对氯福克酚的抵抗可能是由于其不能通过革兰氏阴性菌外膜的原因。其给药方式为口服及直肠栓剂(1.5g/人，每日，主要给药方式)置肛给药的方式。临床观察结果显示，该药物的副作用不大，但在该药物使用过程中，部分患者会出现斑状丘疹或直肠扩张等副作用。目前对于氯福克酚的报道，主要集中于其对革兰氏阳性菌的抑制，及在上呼吸道感染后的治疗。而在2014年有一篇文献由苏州大学和约翰霍普金斯医学院合作的文章报道了氯福克酚可以通过诱导细胞内质网应激、抑制细胞蛋白翻译及细胞周期阻滞等发挥对前列腺癌的抑制作用，如此揭示了氯福克酚的抑瘤作用。
本发明发现相对于胶质瘤细胞株、人正常星形胶质细胞株、人胚肾细胞，人神经干细胞，氯福克酚在细胞水平对实验室分离得到的四株胶质瘤干细胞株具有特异性抑制效果。在此基础上，本发明从细胞水平验证了氯福克酚可特异性抑制胶质瘤干细胞的自我更新能力，肿瘤球形成能力；并通过斑马鱼、裸鼠胶质瘤移植模型验证了氯福克酚对胶质瘤的治疗效果。由此，氯福克酚将很有可能成为神经胶质瘤的治疗提供新的有力治疗药物。
发明内容
基于上述目的，本发明提供了一种氯福克酚用于制备治疗人神经胶质瘤的药物的用途。
在一个具体的实施方案中，所述药物通过抑制胶质瘤干细胞的细胞活性以治疗所述人神经胶质瘤。
在一个优选的实施方案中，所述药物通过抑制胶质瘤干细胞的自我更新、肿瘤球形成，及体内肿瘤形成的能力以治疗所述人神经胶质瘤。
在一个优选的实施方案中，所述药物通过诱导胶质瘤干细胞凋亡以治疗所述人神经胶质瘤。
在一个可选的实施方案中，所述胶质瘤干细胞包括但不限于：U87MG SLC、U251SLC、GSC2、GSC5。
在一个可选的实施方案中，所述药物还通过抑制胶质瘤细胞以治疗所述人神经胶质瘤。
在一个可选的实施方案中，所述胶质瘤细胞包括但不限于：U251、U87MG、N3。
在一个可选的实施方案中，所述人神经胶质瘤处于I到IV级。
术语的定义
“胶质瘤干细胞”：存在于胶质瘤组织内存在的一类极少数的具有自我更新能力，多项分化能力，体内成瘤能力的细胞，它与胶质瘤的发生发展，复发以及胶质瘤对放疗化疗抗性形成密切相关。
“MTS方法”：一种用比色法来检测细胞增殖和细胞毒实验中的活细胞数量的检测试剂。MTS是一种新型四唑化合物，PMS是一种电子偶联剂。PMS具有增强的化学稳定性，这使它可与MTS混合形成稳定的溶液。MTS被细胞生物还原成为一种有色的甲臜产物，可直接溶解于培养基中。这种转化很可能是在代谢活跃的细胞中的脱氢酶产生的NADPH或NADH的作用下完成的。在490nm处检测到的甲臜产物的量与培养中的活细胞数成正比。
“相对细胞存活率”：运用据上MTS方法检测吸光度值后，根据吸光值A计算相对细胞存活率，相对细胞存活率＝(实验组A/对照组A)×100％。
“自我更新”：指干细胞(此处特指胶质瘤干细胞)通过对称或者不对称分裂 产生至少一个保留干细胞特性子细胞的过程，自我更新能够维持干细胞具有多分化的潜能，对于组织特异性干细胞而言，自我更新是维持其终生具有分化潜能的基础。
“肿瘤球形成能力”：指肿瘤干细胞(此处特指胶质瘤干细胞)在体外含EGF，bFGF的培养基中培养，会形成肿瘤球的能力。
综上，本发明发现临床药物氯福克酚可用于制备治疗人神经胶质瘤的药物，从而为人神经胶质瘤的治疗提供新的有力药物，具有显著的社会效益和经济效益。
附图说明
图1：氯福克酚特异性抑制胶质瘤干细胞的细胞活性。MTS(四唑化物)方法检测氯福克酚在不同浓度(0.3μM、1μM、3μM、10μM、30μM)下对四株胶质瘤干细胞株(U87MG SLC、U251SLC、GSC2、GSC5)，三株胶质瘤细胞株(U251、U87MG、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞))、三种人正常细胞株(人正常星形胶质细胞(HA)、人神经干细胞(H1p56NSC)、人胚肾细胞(293ET))作用48小时后细胞存活率，“——”表示IC50值>106μM。
图2A和图2B：氯福克酚特异性抑制胶质瘤干细胞。将带由绿色荧光标记的GSC2-GFP(图2A)或U87MG SLC-GFP(图2B)与不带荧光标记的HA 1:1混匀于体系中，而后氯福克酚分别在1μM、3μM、10μM、30μM浓度下作用24小时，而后流式细胞仪分析不同处理组下存活细胞中GSC2-GFP或U87MG SLC-GFP及HA的比例，并结合共培养模型在不同浓度、不同作用时间下的细胞存活率，统计分析共培养模型中各细胞成分的相对细胞存活率。
图3A至图3H：氯福克酚抑制胶质瘤干细胞肿瘤球的生长与维持。96孔细胞培养皿中每孔加入100μL含5000个GSC2的单细胞悬浮液，自然生长4天后，吸去原培养基，每孔加入含各浓度药物的100μL新鲜培养基，并使其药物浓度分别为：0μM(对照，图3A)、0.03μM(图3B)、0.1μM(图3C)、0.3μM(图3D)、1μM(图3E)、3μM(图3F)、10μM(图3G)、30μM(图3H)，药物作用时间为48小时，利用体外实验模拟临床上胶质瘤干细胞形成肿瘤后检测氯福克酚的抑制效果。氯福克酚可显著减小肿瘤球的体积、数量，且低浓度(0.03μM、0.1μM)GSC2可长出突起，呈现明显的分化样特征，高浓度下(10μM、30μM)，可使肿瘤球边缘细胞死亡并直至整个肿瘤球；图3A至图3H为光镜下检测形态改变。
图4：图3A至图3H中实验的数据统计结果。
图5：氯福克酚抑制胶质瘤干细胞的自我更新能力。将GSC5细胞稀释为25、50、100、200个/100μL，氯福克酚在1μM、3μM、10μM、30μM浓度下处理2周，而后统计无肿瘤球孔百分比，纵坐标表示无肿瘤球孔百分比的对数值。
图6A和图6B：氯福克酚对胶质瘤干细胞的作用效果一定程度上是不可逆的。药物处理GSC2 24小时，48小时后分别撤药物，换上新鲜培养基继续培养48小 时后利用MTS的方法检测各处理组细胞存活率，并比较其与一直用药物处理组的细胞活性改变。“1天”、“2天”表示药物处理24小时或48小时后MTS检测；“药物继续作用”表示药物处理24小时、48小时后撤药物，并继续加入相应药物浓度的新鲜培养基培养48小时后MTS检测；“撤去药物”表示药物处理24小时、48小时后撤药物，并加入不含药物的新鲜培养基继续培养48小时后MTS检测。
图7A和图7B：氯福克酚在30μM浓度下，可明显抑制其裸鼠原位肿瘤球形成能力。氯福克酚在30μM浓度下对GSC2预处理时间24小时，而后收集活细胞，取部分细胞台盼蓝染色后细胞计数，细胞颅内成瘤，105个活细胞/只(5只裸鼠)，并设置DMSO对照组(5只裸鼠)，DMSO处理组(图7A)，及氯福克酚预处理组(图7B)裸鼠原位成瘤后HE染色结果。
图8A和图8B：氯福克酚可显著抑制斑马鱼胶质瘤移植瘤模型的肿瘤生长。48小时斑马鱼胚胎的卵黄囊内注射3000个带有绿色荧光标记的U87MGSLC-GFP，自然生长48小时后，氯福克酚10μM浓度下对其治疗48小时(图8B)，荧光显微镜检测斑马鱼卵黄囊部位绿色荧光的强弱，用以判断氯福克酚对斑马鱼移植瘤模型的抑瘤效果，图8A为对照组。
图9A至图9C：氯福克酚可显著抑制斑裸鼠移植瘤模型的肿瘤生长，且副作用较小。将GSC2细胞接种于15只5周大的Balb/C裸鼠腋下皮下，接种数目为105个/只，当裸鼠皮下肿瘤长至约100mm3，将其分为DMSO组、及氯福克酚药物给药组，给药剂量为20mg/kg，腹腔注射给药，给药时间为11天，期间记录两组裸鼠肿瘤的体积(图9A和图9B)及体重(图9C)变化。
图10A-1至图10A-5：氯福克酚可诱导胶质瘤干细胞凋亡。氯福克酚在0μM(对照，图10A-1)、1μM(图10A-2)、3μM(图10A-3)、10μM(图10A-4)、30μM(图10A-5)浓度下处理GSC2 12小时后，AnnexinⅤ/PI双染，流式分析各处理组内细胞的凋亡情况，图10A-1至图10A-5中(门：P2)发现氯福克酚可明显诱导胶质瘤干细胞的凋亡。
图10B-1至图10B-5：氯福克酚可诱导胶质瘤干细胞凋亡。氯福克酚在0μM(对照，图10A-1)、1μM(图10A-2)、3μM(图10A-3)、10μM(图10A-4)、30μM(图10A-5)浓度下处理24小时后，AnnexinⅤ/PI双染，流式分析各处理组内细胞的凋亡情况，图10B-1至图10B-5中(门：P2)发现氯福克酚可明显诱导胶质瘤干细胞的凋亡。
具体实施方式
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说 明，均可容易地从商业公司获取。
实施例1：MTS法检测细胞存活率
(1)按照产品说明书配制以下溶液：
a、DPBS：依次加入0.2gKCl，8.0gNaCl，0.2gKH2PO4，1.15gNa2HPO4至1L，调pH至7.35，之后加入0.1gMgCl2.6H2O，充分混匀至溶液清澈后，加入0.133gCaCl2.2H2O后，在此充分混匀至溶液清澈。用0.2μM滤器过滤除菌两次后，分装保存在4℃；
b、PMS溶液的配制：使用DPBS配制0.92mg/mL的PMS溶液，用0.2μM滤器过滤除菌两次后，分装于用锡泊纸包裹好的EP管中，避光保存-20℃；
c、MTS溶液的配制：按照1mg MTS粉末用0.5mL DPBS来溶解的比例配制，轻轻混匀，约15min，至MTS完全溶解。测量pH，使其保存在6.0-6.5之间。用0.2μM滤器过滤除菌两次后，分装于用锡泊纸包裹好的EP管中，避光保存-20℃；
d、MTS/PMS溶液的配制：分别融化MTS和PMS，37℃孵育15min，之后按照4.2mg MTS粉末溶于2.1mL DPBS中，使用时加入0.1mL PMS的比例来配制。
(2)对于氯福克酚对胶质瘤干细胞细胞活性的影响检测，实验用96孔细胞培养板用100ug/mL多聚赖氨酸包被过夜，次日生理盐水洗两遍，晾干后待用。将细胞消化后重悬为单细胞悬液，细胞计数，铺于96孔细胞培养板内(U87MG SLC(5000个/孔)、U251SLC(10000个/孔)、GSC2(10000个/孔)、GSC5(10000个/孔))，生长过夜。次日加入氯福克酚(购自美国MicroSource公司)，其化学式如式I所示，氯福克酚化学分子式为C21H26Cl2O，结构简式为Clc1cc(Cl)ccc1Cc2cc(ccc2O)C(C)(C)CC(C)(C)C，其系统名称为2-[(2,4-dichlorophenyl)methyl]-4-(2,4,4-trimethylpentan-2-yl)phenol。并设置如下浓度梯度：0.3μM、1μM、3μM、10μM、30μM，每个药物浓度3个复孔，作用48小时。
Figure PCTCN2017077346-appb-000001
(3)对于三株胶质瘤细胞株(U251(购自美国ATCC)、U87MG(购自美国ATCC)、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞，获赠于天坛医院江涛老师实验室))、三种人正常细胞株(人正常星形胶质细胞(HA，购自美国ATCC)、人神经干细胞(H1p56NSC，本实验室建立的细胞株)、人胚肾细胞(293ET，购自中国医学科学院基础医学研究所细胞中心))则无需包被实验用96孔细胞进行包被。其细胞铺板浓度均为：5000个/孔。其它操作如上。
(4)氯福克酚作用48小时后，根据需要配制相应体积的MTS/PMS检测液， 然后按照检测液：培养基＝1:5的比例加入无血清培养基。最终每孔体系120μL，37℃，5％CO2孵育2小时。根据MTS还原后产生的甲臜产物的吸光度光谱，在吸收峰490nm读取数据。并减去630nm所读取的数据，用以减去细胞碎片等造成的噪音值，以及其它非特异性吸光度值。
(5)根据吸光值A计算细胞存活率，细胞存活率＝(实验组A/对照组)×100％。计算IC50值，并绘制剂量反应曲线。
结果见图1，氯福克酚特异性抑制胶质瘤干细胞的细胞活性。
实施例2：真核细胞慢病毒感染
具体步骤如下：
(1)感染前将GSC2、U87MG SLC、HA细胞消化为单细胞悬液，计数后以相应密度(1-3×105个/mL)接种于6孔细胞培养板内，体系为2mL，让GSC2、U87MG SLC自然生长2小时，HA细胞自然生长直至细胞贴壁(大约4小时)；
(2)分别往GSC2细胞培养基中加入带有绿色荧光的含LV3载体(带嘌呤霉素筛选标记)，往U87MG SLC细胞培养基中加入带有绿色荧光的含pLenti6载体带杀稻瘟素(Blasticidin)筛选标记，购自Promega公司)的慢病毒2μL，往HA细胞培养基中加入带有红色荧光的慢病毒(不带筛选标记)2μL，充分混匀，过夜培养；
(3)次日上午，移去含病毒培养基，各加入新鲜培养基2mL，让其自然生长；
(4)48小时～72小时，荧光显微镜下观察细胞，检测是否有相应荧光表达，以确定是否感染成功；
(5)确定有荧光表达后，用相应浓度的杀稻瘟素筛选稳定表达绿色荧光的U87MG SLC细胞，用相应浓度的嘌呤霉素筛选稳定表达绿色荧光的GSC2细胞，并通过挑选单克隆的方法筛选稳定表达红色荧光的HA细胞。
实施例3：流式细胞分析
预先用100μg/mL的多聚赖氨酸包被6孔及96孔细胞培养板，37℃过夜，生理盐水洗两遍，晾干。将稳定表达绿色荧光的GSC2-GFP细胞及稳定表达红色荧光的HA细胞，消化为单细胞，新鲜培养基重悬，细胞计数，均稀释为5×104个/mL，两种细胞1:1充分混匀，分为五管，分别加入氯福克酚，混匀，使其浓度分别为0μM、1μM、3μM、10μM、30μM。分别加入6孔细胞培养板(2mL)及96孔细胞培养板(100μL，每组3个复孔)，分别待其作用24小时，MTS方法检测96孔细胞培养板中各处理组细胞活性，并分别收集6孔细胞培养板中细胞，PBS洗两遍，各用100μL重悬细胞，流式分析各处理组活细胞中GSC2-GFP、HA-RFP所占比例。并结合共培养模型经药物处理后细胞活性变化，统计分析共培养模型中各细胞成分的相对细胞存活率。共培养模型——U87MG SLC-GFP+HA-RFP的操作如上；
结果见图2A、图2B，相对于人正常星形胶质细胞，氯福克酚可特异性的抑制共培养模型中胶质瘤干细胞的细胞活性。
实施例4：肿瘤球形成实验
96孔细胞培养皿包被后每孔加入100μL含5000个GSC2单细胞悬浮液，自然生长4天后，吸去原培养基，每孔加入各浓度氯福克酚的100μL新鲜培养基，药物浓度分别为：0.03μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM，药物作用时间为处理48小时后镜下观察各处理组肿瘤球的形态变化，并同时统计各处理组肿瘤球数目；
结果见图3A至图3H和图4，利用该体外实验模拟临床上胶质瘤干细胞形成肿瘤后检测氯福克酚的抑制效果，发现氯福克酚可显著减小肿瘤球的体积、数量，且低浓度(0.03μM、0.1μM)GSC2可长出突起，呈现明显的分化样特征，高浓度下(10μM、30μM)，可使肿瘤球边缘细胞死亡并直至整个肿瘤球。
实施例5：有限稀释实验
步骤如下：
(1)将悬浮培养的GSC5肿瘤球细胞收集到15mL离心管中，加入0.5mLACCUTASE酶(购自sigma公司)37℃消化5mins，将细胞吹成单细胞，离心去上清；
(2)加入新鲜的Neurobasal培养基(购自gibco公司)，并取部分细胞台盼蓝染色后细胞计数；
(3)计算所需细胞液的体积，设置细胞浓度梯度，200，100，50，25个/孔，每种浓度设置10个重复孔。设置DMSO对照组，及氯福克酚0μM、1μM、3μM、10μM药物处理组；
(4)将细胞接种于96孔板中，100μL/孔，37℃，5％CO2，孵箱中培养，2周后统计无肿瘤球孔的比例；
结果见图5，氯福克酚在1μM、3μM、10μM浓度下可显著抑制胶质瘤干细胞克隆形成，即能明显抑制胶质瘤干细胞的自我更新能力。
实施例6：回复试验(Recovery assay)
(1)用100ug/mL多聚赖氨酸包被96孔细胞培养板
(2)ACCUTASE酶将GSC5消化为单细胞，新鲜Neurobasal培养基重悬，将细胞铺于预先包被的96孔细胞培养板，过夜；
(3)加入氯福克酚，使其浓度为0.03μM、0.1μM、0.3μM、1μM、3μM、10μM、30μM，每个浓度9个复孔；
(4)作用24小时后，往每个浓度的3个复孔中加入20μL MTS/PMS混合试 剂，检测其细胞活性。吸去其它6个复孔加药培养基，往其中3个孔中加入新鲜培养基，另外3个复孔中加入相同浓度的含氯福克酚培养基；
(5)继续培养48小时后，MTS检测细胞活性。
药物作用48小时，而后检测恢复48小时的情况，操作如上。
结果见图6A和图6B，氯福克酚对胶质瘤干细胞的作用效果一定程度上是不可逆的。
实施例7：裸鼠原位成瘤
收集经氯福克酚30μM度下预处理24小时后细胞，PBS洗两遍，部分细胞台盼蓝染色，细胞计数。取细胞原位注射于5～7周龄Balb/C裸鼠颅内纹状体，105个细胞/只，每组5只，并设置DMSO对照组。
结果见表1、图7A和图7B，氯福克酚预处理胶质瘤干细胞GSC2，可显著抑制其裸鼠原位成瘤能力。
表1
#细胞(GSC2) 对照 氯福克酚-30μM
1*105 5/5 0/5
实施例8：斑马鱼胶质瘤移植模型给药实验
往48小时斑马鱼胚胎的卵黄囊内注射3000个带有绿色荧光标记的U87MGSLC-GFP，让其自然生长48小时，利用荧光显微镜检测斑马鱼体内肿瘤形成情况，而后往斑马鱼孵化液中加入氯福克酚，使其浓度为10μM，药物处理48小时，荧光显微镜检测斑马鱼卵黄囊部位绿色荧光的强弱，用以判断氯福克酚对斑马鱼移植瘤模型体内肿瘤的抑制效果。
结果见图8A和图8B，氯福克酚可显著抑制斑马鱼移植瘤模型体内肿瘤的生长。
实施例9：
裸鼠腋下皮下成瘤：将离心消化后的重悬于PBS的GSC2单细胞接种于15只5周大的Balb/C裸鼠腋下皮下，接种数目为105个/只，记录裸鼠的肿瘤大小(裸鼠肿瘤体积＝长*宽2/2)，当裸鼠皮下肿瘤长至约100mm3时，将其分为DMSO组、及氯福克酚药物给药组，给药剂量为20mg/kg，腹腔注射给药，给药时间为11天，期间记录两组裸鼠肿瘤的体积及体重变化。
结果见图9A至9C，氯福克酚可显著抑制斑裸鼠移植瘤模型的肿瘤生长，且其副作用较小。
实施例10：AnnexinⅤ/PI双染检测细胞凋亡
具体步骤如下；
(1)氯福克酚在1μM、3μM、10μM、30μM浓度下处理GSC2 12小时或24小时；
(2)收集细胞，PBS洗两遍；
(3)取50μL 1*结合缓冲液(binding buffer)重悬细胞，使细胞密度约为1*106个/mL；
(4)加入2.5μL FTIC AnnexinⅤ和2.5μL PI；
(5)避光情况下，室温孵育15min；
(6)往体系中加入200μL1*结合缓冲液(binding buffer)，各处理管分别过细胞筛；
(7)流式细胞仪分析；
所用AnnexinⅤ/PI双染试剂盒购自BD pharmingen，产品批号为556547。
结果见图10A-1至图10B-5，氯福克酚可诱导胶质瘤干细胞细胞凋亡。
综上，本发明在细胞水平证实氯福克酚相对于胶质瘤细胞(U251、U87MG、N3(从病人肿瘤组织分离得到的原代胶质瘤细胞))、人正常星形胶质细胞(HA)、人胚肾细胞(293ET)，人神经干细胞(H1P56NSC)对实验室分离得到的四株胶质瘤干细胞(U87MG SLC、U251SLC、GSC2、GSC5)具有明显的特异性抑制效果(48小时作用的IC50值为：7.65～10.53μM)。并进一步通过克隆形成实验，肿瘤球抑制实验，原位成瘤实验等证实氯福克酚可明显抑制胶质瘤干细胞的自我更新，肿瘤球形成及肿瘤形成能力。而后本发明利用斑马鱼胶质瘤移植瘤模型、裸鼠胶质瘤皮下移植瘤模型，在体水平证明了氯福克酚对胶质瘤的治疗作用。且本发明发现氯福克酚明显的诱导胶质瘤干细胞细胞凋亡，在一定程度上解释了氯福克酚发挥对胶质瘤干细胞抑制作用及其发挥对胶质瘤抑瘤效果的机制。
因此，本发明首次发现临床药物氯福克酚可通过抑制人神经胶质瘤干细胞达到治疗人神经胶质瘤的目的。众所周知，小分子化合物在体内发挥功能都需倚靠相关的关键基团，而在此基础上其它基团的改变虽能产生不同的化合物，但也能发挥其目的功能。因此，任何在此基础上对氯福克酚的修饰，修改后达到与本发明相同效果的研究均用于本发明。

Claims (8)

  1. 氯福克酚用于制备治疗人神经胶质瘤的药物的用途。
  2. 根据权利要求1所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过抑制胶质瘤干细胞的细胞活性以治疗所述人神经胶质瘤。
  3. 根据权利要求1所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过抑制胶质瘤干细胞的自我更新、肿瘤球形成，及体内肿瘤形成的能力以治疗所述人神经胶质瘤。
  4. 根据权利要求1所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中所述药物通过诱导胶质瘤干细胞凋亡以治疗所述人神经胶质瘤。
  5. 根据权利要求1至4中任一项所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中，所述胶质瘤干细胞包括但不限于：U87MG SLC、U251SLC、GSC2、GSC5。
  6. 根据权利要求1所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中所述药物还通过抑制胶质瘤细胞以治疗所述人神经胶质瘤。
  7. 根据权利要求6所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中，所述胶质瘤细胞包括但不限于：U251、U87MG、N3。
  8. 根据权利要求1所述的氯福克酚用于制备治疗人神经胶质瘤的药物的用途，其中，所述人神经胶质瘤处于I到IV级。
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