靶点 |
Hematopoietic cytokines; human hematopoietic stem
cell self-renewal
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体外研究 (In Vitro) |
体外活性:UM171 是 UM729 的合成类似物,因其扩增人 CD34+ CD45RA- 动员的外周血细胞的能力而被筛选出来。 UM171 的效力比 UM729 强 10 至 20 倍,有效浓度为 17 至 19 nM。 UM171 处理使 CD34+ 猕猴细胞的植入潜力提高了三倍。还发现UM171对有丝分裂没有直接影响,对表型原始群体的分裂率也没有影响。此外,UM171与StemRegenin (SR1)合作增强短寿命祖细胞的扩增,而UM171本身选择性增强长期HSC。此外,在 NSG 小鼠中,UM171 对通过移植新鲜或扩增细胞获得的人类造血重建中的淋巴缺陷分化产生影响。细胞测定:用35 nM UM171处理细胞(CD34+CB细胞)3、12、24、48和72小时。在每个时间点,裂解细胞,提取 RNA 并测序。 UM171治疗伴随着与红细胞和巨核细胞分化相关的转录物的显着抑制。在暴露于 UM171 的细胞中,只有六到七个基因通常上调或下调。 UM171 处理的细胞中上调程度最高的基因编码表面分子。这些基因包括 PROCR(也称为 EPCR 或 CD201),它代表小鼠 LT-HSC 的已知标记。
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体内研究 (In Vivo) |
NSG 小鼠注射了最初在 DMSO 或 UM171 中培养的 CD34+ CB 细胞。测定了约 300 只小鼠的人类细胞植入水平,并以热图的形式表示。对该数据集的分析表明人类重建的两种新兴模式,一种主要来自于淋巴髓样 LT-HSC,在大多数情况下以高细胞剂量观察到,另一种来自 LT-HSC,其表现出淋巴缺陷分化表型,主要在 UM171 治疗中观察到。 UM171 不会对 B 淋巴细胞生成以及淋巴细胞 LT-HSC 的频率或数量产生负面影响。 UM171 对 LT-HSC 的影响在移植后 30 周得以保留,此时在高细胞剂量下多谱系贡献仍然明显。
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细胞实验 |
初筛和流式细胞术[1]
CD34+mPB细胞在化合物存在下以每孔2000个细胞接种。培养7天后,使用流式细胞术测定CD34+CD45RA-细胞的相对和绝对数量。如图1所示,选择了主要命中。用配备有高通量筛选模块的LSRII流式细胞仪进行初步筛选。细胞在补充有2%胎牛血清(FBS)的PBS中,在4°C下用APC标记的抗人CD34和PE标记的抗人类CD45RA染色15分钟 使用以下抗体和荧光团的组合测量新鲜和扩增的细胞中的细胞表型:FITC标记的抗人CD34、PE标记的抗人类CD45RA、PE-Cy7-标记的抗人体CD38、APC标记的抗人群CD90、PE-Cy 5-标记的抗人员CD49f和APC标记的反人类EPCR。将染色的细胞用补充有2%FBS的PBS洗涤一次并进行分析。 |
动物实验 |
Transplantation and monitoring of human CD34+ CB cells in NSG mice [1]
All experiments with animals were conducted under protocols approved by the Animal Care Committee of Université de Montréal. Fresh CD34+ CB cells or their progeny present in 12-day cultures were transplanted by tail vein injection into sub-lethally irradiated (250 cGy, <24 hr before transplantation) 8 to 12 week-old female NSG (NODScid IL2Rnull, Jackson Laboratory). Human cells in NSG bone marrow (BM) was monitored by flow cytometry 20 and 30 weeks post-transplantation. NSG BM cells were collected by femoral aspiration (at week 20) or by flushing the two femurs, tibias and hips when animals were sacrificed at week 30. For secondary transplants, 25,000 freshly isolated (uncultured) CD34+ CB cells or the progeny of the equivalent of 10,000 freshly isolated CD34+ CB cells that had been originally cultured in DMSO or UM171 were injected into secondary sub-lethally irradiated NSG mice. BM cells of the secondary mice were harvested and analyzed 18 weeks post-transplantation. Flow cytometry analysis was performed on freshly collected BM cells. Cells were treated with 1x red blood cell lysis buffer, washed and stained with pacific blue-labelled anti-human CD45, APC-eFluo-labelled 780 anti-mouse CD45, PE-labelled anti-human CD33, PE-Cy5 labelled antihuman CD11b, FITC-labelled anti-human CD15, APC-Cy7 labelled anti-human CD14, PE-Cy7 labelled anti-human CD19, FITC-labelled anti-human CD3, APC-labelled antihuman CD71, PE-labelled anti-human glycophorin A (GPA), FITC-labelled anti-human CD41. Cells then were washed and analyzed using a FACSCanto II. BD FACSDiva or FlowJo software were used to analyze the flow cytometry data. The correspondence between antibody labeling and cell populations is as follows: CD11b, CD14, CD15, CD33 (monocytes and granulocytes); CD56 (NK cells); CD71, GPA, CD41 (erythroid and megakaryocyte lineages); CD19, CD3 (B and T cells) and CD34 (stem/progenitor cells). A total of 300,000 BM cells were analyzed per mouse. Considering current FACS limit of detection of 1 per 20,000 events, a minimum threshold of 15 human cells could be detected in these mice. Because some lymphoid cells are long-lived, their presence after a long time can be potentially misleading for assessing LT-HSC activity, which is more conservatively assessed by relying on detecting the sustained output of short-lived cells (e.g., granulocytes).To detect human LT-HSC contribution to NSG reconstitution, we arbitrarily set the lower limit of human cell engraftment at 10-times the FACS threshold or 150 myeloid cells per 300,000 BM cells. T-cell reconstitution was not monitored because it correlates poorly with LT-HSC engraftment in other studies (5) and may represent graft versus host disease, a state that could be associated with peripheral amplification of T-cells not reflecting LT-HSC activity. Similarly, low levels of B-cell restricted reconstitution (<0.05%) was not considered for LT-HSC evaluation because of the extended longevity of B lymphocytes in vivo. However, these criteria led to the reclassification of only 5 mice in our cohort of 300. |
参考文献 |
[1]. Science.2014 Sep 19;345(6203):1509-12.
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其他信息 |
The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.[1]
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分子式 |
C25H27N9
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分子量 |
453.54
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精确质量 |
453.238
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CAS号 |
1448724-09-1
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相关CAS号 |
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PubChem CID |
71714981
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外观&性状 |
Off-white to yellow solid
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密度 |
1.5±0.1 g/cm3
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沸点 |
697.7±65.0 °C at 760 mmHg
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闪点 |
375.8±34.3 °C
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蒸汽压 |
0.0±2.2 mmHg at 25°C
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折射率 |
1.799
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LogP |
3.34
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tPSA |
123Ų
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氢键供体(HBD)数目 |
3
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氢键受体(HBA)数目 |
7
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可旋转键数目(RBC) |
5
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重原子数目 |
34
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分子复杂度/Complexity |
665
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定义原子立体中心数目 |
0
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SMILES |
CN1N=C(C2=CC3=C(C=C2)C4=C(N[C@H]5CC[C@@H](CC5)N)N=C(CC6=CC=CC=C6)NC4=N3)N=N1
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InChi Key |
AZXXGVPWWKWGAE-IYARVYRRSA-N
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InChi Code |
InChI=1S/C25H27N9/c1-34-32-23(31-33-34)16-7-12-19-20(14-16)28-25-22(19)24(27-18-10-8-17(26)9-11-18)29-21(30-25)13-15-5-3-2-4-6-15/h2-7,12,14,17-18H,8-11,13,26H2,1H3,(H2,27,28,29,30)/t17-,18-
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化学名 |
(1r,4r)-N1-(2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine
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别名 |
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HS Tariff Code |
2934.99.9001
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存储方式 |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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运输条件 |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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溶解度 (体外实验) |
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溶解度 (体内实验) |
注意: 如下所列的是一些常用的体内动物实验溶解配方,主要用于溶解难溶或不溶于水的产品(水溶度<1 mg/mL)。 建议您先取少量样品进行尝试,如该配方可行,再根据实验需求增加样品量。
注射用配方
注射用配方1: DMSO : Tween 80: Saline = 10 : 5 : 85 (如: 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)(IP/IV/IM/SC等) *生理盐水/Saline的制备:将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中,得到澄清溶液。 注射用配方 2: DMSO : PEG300 :Tween 80 : Saline = 10 : 40 : 5 : 45 (如: 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline) 注射用配方 3: DMSO : Corn oil = 10 : 90 (如: 100 μL DMSO → 900 μL Corn oil) 示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液,加到 900 μL Corn oil/玉米油中, 混合均匀。 View More
注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如:100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)] 口服配方
口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠) 口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素) 示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中,得到0.5%CMC-Na澄清溶液;然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中,得到悬浮液。 View More
口服配方 3: 溶解于 PEG400 (聚乙二醇400) 请根据您的实验动物和给药方式选择适当的溶解配方/方案: 1、请先配制澄清的储备液(如:用DMSO配置50 或 100 mg/mL母液(储备液)); 2、取适量母液,按从左到右的顺序依次添加助溶剂,澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明,并不一定代表此产品 的实际溶解配方): 10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline); 假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中,混合均匀/澄清;向上述体系中加入50 μL Tween-80,混合均匀/澄清;然后继续加入450 μL ddH2O (或 saline)定容至 1 mL; 3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例; 4、 如产品在配制过程中出现沉淀/析出,可通过加热(≤50℃)或超声的方式助溶; 5、为保证最佳实验结果,工作液请现配现用! 6、如不确定怎么将母液配置成体内动物实验的工作液,请查看说明书或联系我们; 7、 以上所有助溶剂都可在 Invivochem.cn网站购买。 |
制备储备液 | 1 mg | 5 mg | 10 mg | |
1 mM | 2.2049 mL | 11.0244 mL | 22.0488 mL | |
5 mM | 0.4410 mL | 2.2049 mL | 4.4098 mL | |
10 mM | 0.2205 mL | 1.1024 mL | 2.2049 mL |
1、根据实验需要选择合适的溶剂配制储备液 (母液):对于大多数产品,InvivoChem推荐用DMSO配置母液 (比如:5、10、20mM或者10、20、50 mg/mL浓度),个别水溶性高的产品可直接溶于水。产品在DMSO 、水或其他溶剂中的具体溶解度详见上”溶解度 (体外)”部分;
2、如果您找不到您想要的溶解度信息,或者很难将产品溶解在溶液中,请联系我们;
3、建议使用下列计算器进行相关计算(摩尔浓度计算器、稀释计算器、分子量计算器、重组计算器等);
4、母液配好之后,将其分装到常规用量,并储存在-20°C或-80°C,尽量减少反复冻融循环。
计算结果:
工作液浓度: mg/mL;
DMSO母液配制方法: mg 药物溶于 μL DMSO溶液(母液浓度 mg/mL)。如该浓度超过该批次药物DMSO溶解度,请首先与我们联系。
体内配方配制方法:取 μL DMSO母液,加入 μL PEG300,混匀澄清后加入μL Tween 80,混匀澄清后加入 μL ddH2O,混匀澄清。
(1) 请确保溶液澄清之后,再加入下一种溶剂 (助溶剂) 。可利用涡旋、超声或水浴加热等方法助溶;
(2) 一定要按顺序加入溶剂 (助溶剂) 。