Dickkopf1在多发性骨髓瘤中的作用
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摘要
第一部分DKK1在多发性骨髓瘤中的临床研究
目的研究MM患者骨髓上清液及血清Dickkopf1(DKK1)水平,及其与骨髓瘤分期、溶骨性病变的关系,以及不同化疗方案对DKK1的影响及与疗效的关系,探讨DKK1在多发性骨髓瘤中的临床作用。
方法采用双抗体夹心酶标免疫分析法(ELISA)检测80例初诊MM患者、10例MGUS患者及20例对照骨髓上清液及血清DKK1水平,以及检测136例接受硼替佐米+地塞米松或沙利度胺+地塞米松治疗的MM患者化疗前后血清DKK1水平。
结果MM患者与MGUS患者相比,骨髓上清液DKK1水平明显增高(9.3588ng/ml vs 1.7620ng/ml,P=0.001)。而MGUS患者与对照组相比,DKK1水平无明显变化(1.7620ng/ml vs 1.2491ng/ml,P>0.05)。骨髓上清液组与血清组之间DKK1水平无统计学差异(9.3588ng/ml vs 9.2437ng/ml,P>0.05)。DKK1水平与骨髓瘤分期(Durie and Salmon分期)相关,II期和III期MM患者骨髓上清液DKK1水平明显高于I期患者(I期2.4410ng/ml vs II、III期10.3471ng/ml,P=0.002)。I期MM患者与MGUS患者骨髓上清液DKK1水平无统计学差异(P>0.05)。使用ISS分期也得到相似结果。重要的是,有溶骨性病变的MM患者骨髓上清液DKK1水平明显高于没有溶骨性病变的患者(11.2272ng/ml vs 2.4348ng/ml,P<0.001)。而且骨髓上清液DKK1水平还与骨损害的病灶数相关(0,1-3,>3,分别为2.4348ng/ml,4.9845ng/ml,15.3342ng/ml,P<0.001)。沙利度胺+地塞米松(TD)组患者化疗前后血清DKK1水平无明显变化(10.5614ng/ml vs 9.5326ng/ml,P>0.05)。硼替佐米+地塞米松(VD)组患者化疗前后血清DKK1水平明显下降(14.1789ng/ml vs8.3972ng/ml,P<0.05)。所有化疗有效(CR+PR)MM患者,化疗前后血清DKK1水平明显下降(11.6838ng/ml vs 7.1931ng/ml,P<0.05);而化疗无效(NC+PD)者,则血清DKK1水平无明显变化(14.0947ng/ml vs 12.1185ng/ml,P>0.05)。在TD组,无论是化疗有效还是无效,化疗前后血清DKK1水平均无明显变化(P>0.05)。VD组化疗有效者,化疗前后血清DKK1水平显著下降(13.3492ng/ml vs 5.9769ng/ml,P<0.01)。VD组化疗无效者,化疗前后血清DKK1水平有所下降,但无统计学差异(16.0644ng/ml vs 13.8978ng/ml ,P>0.05)。
结论MM患者DKK1水平明显高于MGUS患者及对照组,而且DKK1水平与骨髓瘤分期及溶骨性病变密切相关,能反映溶骨性病变的程度和范围。DKK1水平在化疗有效时明显下降,但与化疗方案有关,硼替佐米能使DKK1水平明显下降,而沙利度胺及地塞米松却无此作用。
第二部分骨髓瘤细胞与基质细胞DKK1相关受体LRP5/6及Kremen1/2基因转录水平的研究
目的比较骨髓瘤细胞系、CD138+原代骨髓瘤细胞、MM患者及正常人基质细胞LRP5/6、Kremen1/2及β-catenin mRNA水平,研究骨髓瘤细胞或重组DKK1蛋白对正常人基质细胞上述基因mRNA水平的影响,以探讨DKK1在多发性骨髓瘤中的作用。
方法采用Western blot及ELISA方法,检测骨髓瘤细胞系、CD138+原代骨髓瘤细胞、骨髓基质细胞及细胞培养液DKK1蛋白表达情况,以及Realtime-PCR方法,检测骨髓瘤细胞系、CD138+原代骨髓瘤细胞、MM患者及正常人骨髓基质细胞LRP5/6、Kremen1/2及β-catenin mRNA水平以及骨髓瘤细胞或重组DKK1蛋白与正常人骨髓基质细胞共培养时上述基因mRNA水平。
结果骨髓瘤细胞系RPMI-8226、NCI-H929和LP-1以及3/5例MM患者CD138+骨髓瘤细胞,DKK1蛋白表达明显,而4例MM患者基质细胞中未见DKK1蛋白表达,同时DKK1+骨髓瘤细胞培养液中也检测到DKK1蛋白。MM患者基质细胞LRP5/6和Kremen1/2 mRNA水平显著高于正常人基质细胞和CD138+原代骨髓瘤细胞。CD138+原代骨髓瘤细胞β-catenin mRNA水平高于MM患者及正常人基质细胞。无论使用transwell小室或者直接接触法进行骨髓瘤细胞系RPMI-8226、NCI-H929与正常人基质细胞共培养48h及72h,LRP5/6、Kremen1/2 mRNA水平明显增加,β-catenin mRNA水平下降,但24h变化不明显。72h与48h相比,上述基因mRNA水平变化更加明显。重组DKK1蛋白浓度为100ng/ml及300ng/ml时,与基质细胞共培养48h及72h后LRP5/6、Kremen1/2及β-catenin mRNA水平无明显变化。重组DKK1蛋白浓度为500ng/ml时,培养48h LRP5/6 mRNA水平有轻微上升(P>0.05),Kremen1/2 mRNA水平有所上升(P<0.05),及β-catenin mRNA水平略有下降(P>0.05);培养72h LRP5 mRNA水平有所上升(P<0.05),LRP6 mRNA水平无明显变化(P>0.05);Kremen1/2 mRNA水平有所上升(P<0.05),及β-catenin mRNA水平明显下降(P<0.05)。重组DKK1蛋白浓度为700ng/ml时,培养48h及72h LRP5/6、Kremen1/2 mRNA水平明显上升(P<0.05),及β-catenin mRNA水平明显下降(P<0.05)。
结论骨髓瘤细胞、MM患者及正常人基质细胞之间LRP5/6、Kremen1/2及β-catenin基因转录水平存在差异,而骨髓瘤细胞分泌的DKK1可能是造成MM患者基质细胞上述基因变化的重要因素。
第三部分DKK1的真核表达、纯化及功能测定
目的表达和纯化DKK1蛋白,研究骨髓瘤细胞及基质细胞DKK1相关受体表达情况,并探讨DKK1对骨髓瘤细胞及基质细胞效应不同的可能机制。
方法利用脂质体介导pcDNA3.1(-)-DKK1质粒瞬时转染293T细胞,使用western blot方法鉴定上清中的表达蛋白,使用Ni-Agrose His标签蛋白纯化试剂盒,纯化融合His标签的DKK1蛋白。利用流式细胞术(FCM),对CD138+骨髓瘤细胞、MM患者基质细胞及健康志愿者基质细胞表面DKK1相关受体表达情况进行同步分析比较。
结果使用脂质体转染法,成功将pcDNA3.1(-)-DKK1质粒瞬时转染293T细胞,获得表达蛋白。使用特异性抗Dkk1抗体与小鼠抗6×His单克隆抗体行Western Blot鉴定重组蛋白,证实重组蛋白是融和了His标签的Dkk1蛋白。流式细胞术显示,重组DKK1蛋白能与CD138+骨髓瘤细胞、MM患者基质细胞及健康志愿者基质细胞特异性结合。其中健康志愿者基质细胞平均荧光强度(MFI)为20.8±8.4;CD138+骨髓瘤细胞MFI为58.6±17.2;MM患者基质细胞MFI为104.5±32.1。三组之间相比有统计学差异(P<0.05)。
结论脂质体介导的质粒pcDNA3.1(-)-DKK1体外转染效率较高,能表达有活性的DKK1蛋白,可以与骨髓瘤细胞及基质细胞DKK1相关受体特异性结合。骨髓瘤细胞、基质细胞之间DKK1相关受体蛋白表达量不同。
Part I:Clinical study of DKK1 in multiple myeloma
Objective To detect bone marrow plasma and serum concentrations of Dickkopf1(DKK1) in patients with multiple myeloma and perform a correlative analysis between DKK1, myeloma stage and lytic bone disease and evaluate the effect of different treatment regimens for MM on serum DKK1 and the relationship with the response to treatment.
Methods DKK1 bone marrow plasma and serum levels were quantified in 80 newly diagnosed MM patients、10 monoclonal gammopathy of undetermined significance (MGUS) patients and 20 control subjects by ELISA, using a monoclonal anti-DKK1 antibody. And serum samples were collected from 136 patients with MM treated with bortezomib and dexamethasone (n = 72), thalidomide and dexamethasone (n = 64) before and 3 months after initiation of therapy.
Results Bone marrow plasma DKK1 was elevated in MM as compared with MGUS (mean 9.3588ng/ml vs. 1.762ng/ml; P=0.001). There was no difference between MGUS patients and control subjects(1.762ng/ml vs 1.2491ng/ml; P > 0.05). Bone marrow plasma DKK1 levels significantly correlated with myeloma stage according to Durie and Salmon (mean 2.4410ng/ml vs. 10.3471ng/ml in stage I and II ? III, respectively; P = 0.002). There was no difference between MM patients in stage I and MGUS patients(P>0.05). There were similar results using ISS stage. Importantly, myeloma patients without lytic lesions in conventional radiography had significantly lower DKK1 levels than patients with lytic bone disease (mean 2.4348 ng/ml vs. 11.2272ng/ml; P<0.001). Of interest, bone marrow plasma DKK1 correlated with the number of bone lesions (0 vs. 1–3 vs. >3 lesions: 2.4348ng/ml vs. 4.9845ng/ml vs. 15.3342ng/ml; P<0.001). In this study, the effect of different treatment regimens for MM patients on serum DKK1 was evaluated and correlated with the response to treatment in 136 myeloma patients receiving bortezomib and dexamethasone(n=72), thalidomide and dexamethasone(n=64). A significant decrease of DKK1 after therapy was seen in the Bortezomib and dexamethasone groups (mean 14.1789ng/ml vs. 8.3972ng/ml; P < 0.05). Thalidomide and dexamethasone led to a nonsignificant decrease in DKK1 (10.5614ng/ml vs. 9.5326ng/ml; P>0.05). Within all groups, a significant decrease of DKK1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non-responders.
Conclusion Myeloma patients have increased bone marrow plasma DKK1 as compared with patients with MGUS and control subjects. Bone marrow plasma concentrations of DKK1 have close relationship with myeloma stage and lytic bone disease. Serum DKK1 levels decrease in myeloma patients responding to treatment, corelate with the regimen chosen.
Part II: Gene expression of DKK1 receptor LRP5/6 and Kremen1/2 in myeloma cells and stromal cells
Objective To compare the gene expression of LRP5/6、Kremen1/2 andβ-catenin in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers and investigate the effect of multiple myeloma cells or rhDKK1on gene expression in stromal cells of healthy volunteers and identify the role of DKK1 in multiple myeloma.
Methods DKK1 protein expression was checked in human myeloma cell lines, CD138+ primary myeloma cells, stromal cells and myeloma cell condition media using Western Blot analysis and ELISA assay. LRP5/6、Kremen1/2 andβ-catenin mRNA expression in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers were tested by Realtime-PCR. To compare the effect of multiple myeloma cells or rhDKK1 on gene expression in human stromal cells, we did Real-time PCR for LRP5/6、Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers cocultured with multiple myeloma cells or rhDKK1.
Results DKK1 was produced by multiple myeloma cells, not stromal cells. DKK1 was detectable in myeloma cell condition media, as well. LRP5/6 and Kremen1/2 mRNA expression in stromal cells of multiple myeloma patients were higher than that in stromal cells of healthy volunteer and CD138+ primary myeloma cells.β-catenin mRNA expression in CD138+ primary myeloma cells was higher than that in stromal cells of multiple myeloma patients and healthy volunteers. Gene expression of LRP5/6, Kremen1/2 was elevated in stromal cells of healthy volunteers after 48h -72h of coculture with HMCLs RPMI-8226 in the presence or absence of a Transwell insert. A significant inhibitory effect was observed on the expression ofβ-catenin in stromal cells of healthy volunteers after 48h -72h of coculture with multiple myeloma cells. No significant effect was observed on the gene expression after 24h coculture. Similar results were obtained with the HMCLs NCI-H929 in coculture (data not shown). We examined the effect of rhDKK1 on LRP5/6, Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers. The levels of LRP5/6 and Kremen1/2 gene expression in stromal cells of healthy volunteers were highest at 72h post rhDKK1(at concentration 700 ng/mL) treatment. Andβ-catenin mRNA expression in stromal cells of healthy volunteers decreased significantly at the same time.
Conclusion LRP5/6、Kremen1/2 andβ-catenin gene were expressed differently in human myeloma cell lines, CD138+ primary myeloma cells and stromal cells. And DKK1 produced by myloma cells may play an important role in the different gene expression in stromal cells between multiple myeloma patients and healthy volunteers.
Part III: Expression,purification and functional examination of DKK1
Objective To express, purify DKK1, and investigate the binding capacity of its receptor on the cell membrane of myeloma cells and stromal cells.
Methods Vector pcDNA3.1 (-)-DKK1 was transfected into 293T cells with LipofectAMINETM2000 reagent. DKK1protein in the supernatant was identified by western blot. With the Ni-Agrose His protein purification kit, DKK1 protein was purified. Flow cytometry (FCM) was used to study DKK1-bound receptor on the cell membrane of myeloma cells and stromal cells.
Results With LipofectAMINETM2000 reagent, the mammalian expression vector pcDNA3.1 (-)-DKK1 was successfully transfected into 293T cells. In the supernatant, DKK1 protein was collected. Using anti-DKK1 antibody and anti-His antibody, DKK1 fusion protein in the supernatant was identified by western blot. Flow cytometry results showed that DKK1 protein could specifically bind the receptor on the cell membrane of myeloma cells and stromal cells. The mean fluorescence intensity (MFI) of receptor on myeloma cells, stromal cells of the healthy volunteers and myeloma patients were (58.6±17.2), (20.8±8.4), (104.5±32.1), respectively. There were significantly difference among three groups(P<0.05).
Conclusion Using mammalian expression vector, DKK1 protein was obtained and showed a high biological activity and could specifically bind the receptor. The amount of receptor of DKK1 protein specifically binding on the cell membrane of myeloma cells and stromal cells were different.
目的研究MM患者骨髓上清液及血清Dickkopf1(DKK1)水平,及其与骨髓瘤分期、溶骨性病变的关系,以及不同化疗方案对DKK1的影响及与疗效的关系,探讨DKK1在多发性骨髓瘤中的临床作用。
方法采用双抗体夹心酶标免疫分析法(ELISA)检测80例初诊MM患者、10例MGUS患者及20例对照骨髓上清液及血清DKK1水平,以及检测136例接受硼替佐米+地塞米松或沙利度胺+地塞米松治疗的MM患者化疗前后血清DKK1水平。
结果MM患者与MGUS患者相比,骨髓上清液DKK1水平明显增高(9.3588ng/ml vs 1.7620ng/ml,P=0.001)。而MGUS患者与对照组相比,DKK1水平无明显变化(1.7620ng/ml vs 1.2491ng/ml,P>0.05)。骨髓上清液组与血清组之间DKK1水平无统计学差异(9.3588ng/ml vs 9.2437ng/ml,P>0.05)。DKK1水平与骨髓瘤分期(Durie and Salmon分期)相关,II期和III期MM患者骨髓上清液DKK1水平明显高于I期患者(I期2.4410ng/ml vs II、III期10.3471ng/ml,P=0.002)。I期MM患者与MGUS患者骨髓上清液DKK1水平无统计学差异(P>0.05)。使用ISS分期也得到相似结果。重要的是,有溶骨性病变的MM患者骨髓上清液DKK1水平明显高于没有溶骨性病变的患者(11.2272ng/ml vs 2.4348ng/ml,P<0.001)。而且骨髓上清液DKK1水平还与骨损害的病灶数相关(0,1-3,>3,分别为2.4348ng/ml,4.9845ng/ml,15.3342ng/ml,P<0.001)。沙利度胺+地塞米松(TD)组患者化疗前后血清DKK1水平无明显变化(10.5614ng/ml vs 9.5326ng/ml,P>0.05)。硼替佐米+地塞米松(VD)组患者化疗前后血清DKK1水平明显下降(14.1789ng/ml vs8.3972ng/ml,P<0.05)。所有化疗有效(CR+PR)MM患者,化疗前后血清DKK1水平明显下降(11.6838ng/ml vs 7.1931ng/ml,P<0.05);而化疗无效(NC+PD)者,则血清DKK1水平无明显变化(14.0947ng/ml vs 12.1185ng/ml,P>0.05)。在TD组,无论是化疗有效还是无效,化疗前后血清DKK1水平均无明显变化(P>0.05)。VD组化疗有效者,化疗前后血清DKK1水平显著下降(13.3492ng/ml vs 5.9769ng/ml,P<0.01)。VD组化疗无效者,化疗前后血清DKK1水平有所下降,但无统计学差异(16.0644ng/ml vs 13.8978ng/ml ,P>0.05)。
结论MM患者DKK1水平明显高于MGUS患者及对照组,而且DKK1水平与骨髓瘤分期及溶骨性病变密切相关,能反映溶骨性病变的程度和范围。DKK1水平在化疗有效时明显下降,但与化疗方案有关,硼替佐米能使DKK1水平明显下降,而沙利度胺及地塞米松却无此作用。
第二部分骨髓瘤细胞与基质细胞DKK1相关受体LRP5/6及Kremen1/2基因转录水平的研究
目的比较骨髓瘤细胞系、CD138+原代骨髓瘤细胞、MM患者及正常人基质细胞LRP5/6、Kremen1/2及β-catenin mRNA水平,研究骨髓瘤细胞或重组DKK1蛋白对正常人基质细胞上述基因mRNA水平的影响,以探讨DKK1在多发性骨髓瘤中的作用。
方法采用Western blot及ELISA方法,检测骨髓瘤细胞系、CD138+原代骨髓瘤细胞、骨髓基质细胞及细胞培养液DKK1蛋白表达情况,以及Realtime-PCR方法,检测骨髓瘤细胞系、CD138+原代骨髓瘤细胞、MM患者及正常人骨髓基质细胞LRP5/6、Kremen1/2及β-catenin mRNA水平以及骨髓瘤细胞或重组DKK1蛋白与正常人骨髓基质细胞共培养时上述基因mRNA水平。
结果骨髓瘤细胞系RPMI-8226、NCI-H929和LP-1以及3/5例MM患者CD138+骨髓瘤细胞,DKK1蛋白表达明显,而4例MM患者基质细胞中未见DKK1蛋白表达,同时DKK1+骨髓瘤细胞培养液中也检测到DKK1蛋白。MM患者基质细胞LRP5/6和Kremen1/2 mRNA水平显著高于正常人基质细胞和CD138+原代骨髓瘤细胞。CD138+原代骨髓瘤细胞β-catenin mRNA水平高于MM患者及正常人基质细胞。无论使用transwell小室或者直接接触法进行骨髓瘤细胞系RPMI-8226、NCI-H929与正常人基质细胞共培养48h及72h,LRP5/6、Kremen1/2 mRNA水平明显增加,β-catenin mRNA水平下降,但24h变化不明显。72h与48h相比,上述基因mRNA水平变化更加明显。重组DKK1蛋白浓度为100ng/ml及300ng/ml时,与基质细胞共培养48h及72h后LRP5/6、Kremen1/2及β-catenin mRNA水平无明显变化。重组DKK1蛋白浓度为500ng/ml时,培养48h LRP5/6 mRNA水平有轻微上升(P>0.05),Kremen1/2 mRNA水平有所上升(P<0.05),及β-catenin mRNA水平略有下降(P>0.05);培养72h LRP5 mRNA水平有所上升(P<0.05),LRP6 mRNA水平无明显变化(P>0.05);Kremen1/2 mRNA水平有所上升(P<0.05),及β-catenin mRNA水平明显下降(P<0.05)。重组DKK1蛋白浓度为700ng/ml时,培养48h及72h LRP5/6、Kremen1/2 mRNA水平明显上升(P<0.05),及β-catenin mRNA水平明显下降(P<0.05)。
结论骨髓瘤细胞、MM患者及正常人基质细胞之间LRP5/6、Kremen1/2及β-catenin基因转录水平存在差异,而骨髓瘤细胞分泌的DKK1可能是造成MM患者基质细胞上述基因变化的重要因素。
第三部分DKK1的真核表达、纯化及功能测定
目的表达和纯化DKK1蛋白,研究骨髓瘤细胞及基质细胞DKK1相关受体表达情况,并探讨DKK1对骨髓瘤细胞及基质细胞效应不同的可能机制。
方法利用脂质体介导pcDNA3.1(-)-DKK1质粒瞬时转染293T细胞,使用western blot方法鉴定上清中的表达蛋白,使用Ni-Agrose His标签蛋白纯化试剂盒,纯化融合His标签的DKK1蛋白。利用流式细胞术(FCM),对CD138+骨髓瘤细胞、MM患者基质细胞及健康志愿者基质细胞表面DKK1相关受体表达情况进行同步分析比较。
结果使用脂质体转染法,成功将pcDNA3.1(-)-DKK1质粒瞬时转染293T细胞,获得表达蛋白。使用特异性抗Dkk1抗体与小鼠抗6×His单克隆抗体行Western Blot鉴定重组蛋白,证实重组蛋白是融和了His标签的Dkk1蛋白。流式细胞术显示,重组DKK1蛋白能与CD138+骨髓瘤细胞、MM患者基质细胞及健康志愿者基质细胞特异性结合。其中健康志愿者基质细胞平均荧光强度(MFI)为20.8±8.4;CD138+骨髓瘤细胞MFI为58.6±17.2;MM患者基质细胞MFI为104.5±32.1。三组之间相比有统计学差异(P<0.05)。
结论脂质体介导的质粒pcDNA3.1(-)-DKK1体外转染效率较高,能表达有活性的DKK1蛋白,可以与骨髓瘤细胞及基质细胞DKK1相关受体特异性结合。骨髓瘤细胞、基质细胞之间DKK1相关受体蛋白表达量不同。
Part I:Clinical study of DKK1 in multiple myeloma
Objective To detect bone marrow plasma and serum concentrations of Dickkopf1(DKK1) in patients with multiple myeloma and perform a correlative analysis between DKK1, myeloma stage and lytic bone disease and evaluate the effect of different treatment regimens for MM on serum DKK1 and the relationship with the response to treatment.
Methods DKK1 bone marrow plasma and serum levels were quantified in 80 newly diagnosed MM patients、10 monoclonal gammopathy of undetermined significance (MGUS) patients and 20 control subjects by ELISA, using a monoclonal anti-DKK1 antibody. And serum samples were collected from 136 patients with MM treated with bortezomib and dexamethasone (n = 72), thalidomide and dexamethasone (n = 64) before and 3 months after initiation of therapy.
Results Bone marrow plasma DKK1 was elevated in MM as compared with MGUS (mean 9.3588ng/ml vs. 1.762ng/ml; P=0.001). There was no difference between MGUS patients and control subjects(1.762ng/ml vs 1.2491ng/ml; P > 0.05). Bone marrow plasma DKK1 levels significantly correlated with myeloma stage according to Durie and Salmon (mean 2.4410ng/ml vs. 10.3471ng/ml in stage I and II ? III, respectively; P = 0.002). There was no difference between MM patients in stage I and MGUS patients(P>0.05). There were similar results using ISS stage. Importantly, myeloma patients without lytic lesions in conventional radiography had significantly lower DKK1 levels than patients with lytic bone disease (mean 2.4348 ng/ml vs. 11.2272ng/ml; P<0.001). Of interest, bone marrow plasma DKK1 correlated with the number of bone lesions (0 vs. 1–3 vs. >3 lesions: 2.4348ng/ml vs. 4.9845ng/ml vs. 15.3342ng/ml; P<0.001). In this study, the effect of different treatment regimens for MM patients on serum DKK1 was evaluated and correlated with the response to treatment in 136 myeloma patients receiving bortezomib and dexamethasone(n=72), thalidomide and dexamethasone(n=64). A significant decrease of DKK1 after therapy was seen in the Bortezomib and dexamethasone groups (mean 14.1789ng/ml vs. 8.3972ng/ml; P < 0.05). Thalidomide and dexamethasone led to a nonsignificant decrease in DKK1 (10.5614ng/ml vs. 9.5326ng/ml; P>0.05). Within all groups, a significant decrease of DKK1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non-responders.
Conclusion Myeloma patients have increased bone marrow plasma DKK1 as compared with patients with MGUS and control subjects. Bone marrow plasma concentrations of DKK1 have close relationship with myeloma stage and lytic bone disease. Serum DKK1 levels decrease in myeloma patients responding to treatment, corelate with the regimen chosen.
Part II: Gene expression of DKK1 receptor LRP5/6 and Kremen1/2 in myeloma cells and stromal cells
Objective To compare the gene expression of LRP5/6、Kremen1/2 andβ-catenin in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers and investigate the effect of multiple myeloma cells or rhDKK1on gene expression in stromal cells of healthy volunteers and identify the role of DKK1 in multiple myeloma.
Methods DKK1 protein expression was checked in human myeloma cell lines, CD138+ primary myeloma cells, stromal cells and myeloma cell condition media using Western Blot analysis and ELISA assay. LRP5/6、Kremen1/2 andβ-catenin mRNA expression in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers were tested by Realtime-PCR. To compare the effect of multiple myeloma cells or rhDKK1 on gene expression in human stromal cells, we did Real-time PCR for LRP5/6、Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers cocultured with multiple myeloma cells or rhDKK1.
Results DKK1 was produced by multiple myeloma cells, not stromal cells. DKK1 was detectable in myeloma cell condition media, as well. LRP5/6 and Kremen1/2 mRNA expression in stromal cells of multiple myeloma patients were higher than that in stromal cells of healthy volunteer and CD138+ primary myeloma cells.β-catenin mRNA expression in CD138+ primary myeloma cells was higher than that in stromal cells of multiple myeloma patients and healthy volunteers. Gene expression of LRP5/6, Kremen1/2 was elevated in stromal cells of healthy volunteers after 48h -72h of coculture with HMCLs RPMI-8226 in the presence or absence of a Transwell insert. A significant inhibitory effect was observed on the expression ofβ-catenin in stromal cells of healthy volunteers after 48h -72h of coculture with multiple myeloma cells. No significant effect was observed on the gene expression after 24h coculture. Similar results were obtained with the HMCLs NCI-H929 in coculture (data not shown). We examined the effect of rhDKK1 on LRP5/6, Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers. The levels of LRP5/6 and Kremen1/2 gene expression in stromal cells of healthy volunteers were highest at 72h post rhDKK1(at concentration 700 ng/mL) treatment. Andβ-catenin mRNA expression in stromal cells of healthy volunteers decreased significantly at the same time.
Conclusion LRP5/6、Kremen1/2 andβ-catenin gene were expressed differently in human myeloma cell lines, CD138+ primary myeloma cells and stromal cells. And DKK1 produced by myloma cells may play an important role in the different gene expression in stromal cells between multiple myeloma patients and healthy volunteers.
Part III: Expression,purification and functional examination of DKK1
Objective To express, purify DKK1, and investigate the binding capacity of its receptor on the cell membrane of myeloma cells and stromal cells.
Methods Vector pcDNA3.1 (-)-DKK1 was transfected into 293T cells with LipofectAMINETM2000 reagent. DKK1protein in the supernatant was identified by western blot. With the Ni-Agrose His protein purification kit, DKK1 protein was purified. Flow cytometry (FCM) was used to study DKK1-bound receptor on the cell membrane of myeloma cells and stromal cells.
Results With LipofectAMINETM2000 reagent, the mammalian expression vector pcDNA3.1 (-)-DKK1 was successfully transfected into 293T cells. In the supernatant, DKK1 protein was collected. Using anti-DKK1 antibody and anti-His antibody, DKK1 fusion protein in the supernatant was identified by western blot. Flow cytometry results showed that DKK1 protein could specifically bind the receptor on the cell membrane of myeloma cells and stromal cells. The mean fluorescence intensity (MFI) of receptor on myeloma cells, stromal cells of the healthy volunteers and myeloma patients were (58.6±17.2), (20.8±8.4), (104.5±32.1), respectively. There were significantly difference among three groups(P<0.05).
Conclusion Using mammalian expression vector, DKK1 protein was obtained and showed a high biological activity and could specifically bind the receptor. The amount of receptor of DKK1 protein specifically binding on the cell membrane of myeloma cells and stromal cells were different.
引文
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