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. 2020 Oct 20;40(10):1439–1447. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2020.10.09

唑来膦酸盐通过p38 MAPK通路调控高糖条件下破骨细胞分化生成及骨吸收功能

Zoledronate regulates osteoclast differentiation and bone resorption in high glucose through p38 MAPK pathway

蔺 一凡 1, 谷 营营 1, 左 桂福 2, 贾 顺义 1, 梁 永强 1, 戚 孟春 1, 董 伟 1,*
PMCID: PMC7606243  PMID: 33118518

Abstract

目的

探究在高浓度葡萄糖的条件下，唑来膦酸盐(ZOL)对破骨细胞分化及功能的影响，以及p38丝裂原蛋白活化激酶(p38 MAPK)通路在其中的调控机制。

方法

将RAW264.7细胞向破骨细胞方向分化诱导，分为4组：低糖组(5.5 mmol/L葡萄糖)、高糖组(16.5 mmol/L葡萄糖)、低糖+ZOL组(5.5 mmol/L葡萄糖+0.1 μmol ZOL)、高糖+ZOL组(16.5 mmol/L葡萄糖+ 0.1 μmol ZOL)。MTT法测定各组细胞增殖活性，光学显微镜检测RAW264.7细胞的迁移数目，免疫荧光组织化学染色检测各组破骨细胞细胞骨架的清晰程度。增加第5组：高糖+ ZOL + SB203580(16.5 mmol/L葡萄糖+0.1 μmol ZOL +10 μmol SB203580)后，TRAP染色鉴定各组阳性破骨细胞数量；将RAW264.7细胞与牙本质磨片共同培养，扫描电子显微镜测定各组细胞骨吸收陷窝的数量及面积；实时荧光定量PCR(Real-time PCR)及Western blotting检测破骨细胞相关因子的mRNA和蛋白的表达情况。

结果

细胞增殖实验中各组细胞增殖情况无明显差别。高糖条件对RAW264.7细胞的迁移具有促进作用，差异具有统计学意义(P < 0.05)；抑制细胞骨架的清晰程度及破骨细胞封闭区的形成；下调p38 MAPK、p-p38 MAPK、NFATc1、CTSK、TRAP mRNA和蛋白表达情况从而对破骨细胞的分化及功能发挥抑制作用，差异具有统计学意义(P < 0.05)。加入唑来膦酸盐后，RAW264.7细胞迁移受到抑制，差异具有统计学意义(P < 0.05)；细胞骨架的清晰程度进一步下降，破骨细胞封闭区的形成被进一步破坏破；p38 MAPK、p-p38 MAPK、NFATc1、CTSK、TRAP的mRNA和蛋白质表达降低，进一步抑制破骨细胞分化及功能，差异具有统计学意义(P < 0.05)；加入SB203580 后，破骨细胞分化和骨吸收功能受到抑制，p38 MAPK、p-p38 MAPK 表达受到抑制，下游NFATc1、CTSK、TRAP表达降低，差异具有统计学意义(P < 0.05)。

结论

高糖条件抑制破骨细胞的分化生成和骨吸收功能。ZOL通过p38 MAPK信号通路实现了对高糖条件下破骨细胞分化及功能的抑制作用，提示p38 MAPK分子通路可作为唑来膦酸盐调控糖尿病性骨质疏松的新机制。

Keywords: 唑来膦酸盐, 破骨细胞, 高浓度葡萄糖, p38 MAPK

糖尿病是一种慢性代谢性疾病，其病理表现为血糖升高，可导致糖尿病骨质疏松症(DOP)的发生，也可通过抑制骨形成增加骨折风险[1-2]。骨改建是通过复杂机制调控的，破骨细胞是实现骨吸收功能的效应细胞，破骨细胞分化或功能异常会发生骨质疏松症(OP)、骨硬化症等多种骨代谢疾病[3-4]。有研究发现，高糖条件对破骨细胞分化和骨吸收功能具有抑制作用[5]; 高浓度葡萄糖可靶向作用于破骨细胞内的AMPK/mTOR/ULK1信号通路抑制其自吞噬，从而发挥对破骨细胞的分化及功能发挥抑制作用[6]，但高糖条件对破骨细胞生成及功能作用的相关分子学机制尚未完全研究清楚。

双膦酸盐(BPs)是一种稳定的内源性焦磷酸化合物，可显著抑制骨吸收作用，因此临床中常将此药用于治疗骨质疏松症[7-8]。作为双膦酸盐第三代药物，唑来膦酸盐(ZOL)被证实通过抑制核因子-κB配体受体激活剂(RANKL)诱导的NF-κB活化和JNK磷酸化，降低破骨细胞生成相关基因的表达，抑制破骨细胞分化生成[9]。有研究表明，唑来膦酸钠显著下调血管内皮生长因子受体2(VEGFR2)基因的表达，通过VEGF-VEGFR2通路抑制破骨细胞生成[10]。研究发现，ZOL可通过靶向作用于AMPK信号通路实现对高糖条件下破骨细胞分化生成和骨吸收功能的调控作用[11]。然而，双膦酸盐在高糖条件下对破骨细胞的分化生成和骨吸收功能的作用是否受到其他分子机制的调控仍有待于进一步探究。

丝裂原蛋白活化激酶(MAPK)广泛参与了骨相关细胞的细胞内活动。作为MAPK家族成员之一，p38 MAPK信号通路对骨骼发育的调节发挥重要作用[12-14]。p38 MAPK被证实通过抑制破骨细胞前体细胞增殖和分化来调控破骨细胞的分化生成[15]。然而，p38 MAPK分子通路在ZOL对高糖条件下破骨细胞细胞分化和功能影响的研究中是否发挥调控作用，目前国内外在这方面的的研究尚未见报道。因此，本实验通过研究唑来膦酸盐在高糖条件下对破骨细胞分化和功能的影响，探讨其是否受p38 MAPK通路调节，为双膦酸盐治疗糖尿病骨质疏松症的分子学机制提供新的理论依据。

1. 材料和方法

1.1. 实验材料

1.1.1. 细胞

小鼠单核巨噬细胞RAW264.7细胞(中科院上海细胞库)。

1.1.2. 试剂

试剂sRANKL(Biovision); α-MEM培养基(Gibco); 进口胎牛血清(Bovogen); Transwell培养室(Corning); 唑来膦酸盐、MTT、TRAP染色试剂盒(Sigma-Aldrich); SB203580(Selleck); 兔抗小鼠p38 MAPK一抗、p-p38 MAPK一抗(Cell signalling technology); 兔抗小鼠NFATc1、TRAP一抗(Abcam); 兔抗小鼠CTSK一抗(GeneTex); 兔抗小鼠GAPDH一抗(Santa Cruz Biotechnology)。

1.2. 实验方法

1.2.1. 细胞培养和实验分组

用含有破骨细胞诱导因子的培养基(50 ng/mL RANKL、15%胎牛血清、1%双抗的α-MEM)培养基对RAW264.7细胞进行定向诱导分化。实验分组如下：低糖组，培养基内葡萄糖的浓度为5.5 mmol/L; 高糖组，培养基内葡萄糖的浓度为16.5 mmol/L; 低糖+ZOL组，在葡萄糖浓度为5.5 mmol/L的培养基内加入0.1 μmol ZOL; 高糖+ZOL组，在葡萄糖浓度为16.5 mmol/L培养基内加入0.1 μmol ZOL。将将各组细胞放置于温度37 ℃、5% CO2细胞培养箱中，细胞培养基每2 d更换一次。

1.2.2. 细胞活性检测

将各组细胞接种至96孔板中，接种密度：4×103/孔。破骨细胞经诱导分化24、48、72 h后，吸去每孔培养基，加入PBS洗涤3次，向每孔加入20 μL MTT，在37 ℃培养箱孵育4 h后，将上清液液吸去，向每孔中加入150 μL DMSO，在37 ℃摇床中充分摇动孔板10 min，应用酶标仪读取490 nm处的吸收峰，绘制4组细胞的生长曲线图。

1.2.3. 细胞迁移实验

使用Transwell系统对破骨细胞迁移情况进行检测。将RAW264.7细胞以5×104/孔的密度接种于Transwell培养室的上室中，上室的培养基内不加胎牛血清，下室培养基内加入含15%胎牛血清，诱导细胞向下室迁移。细胞诱导迁移24、48 h后，吸去培养基，向下室内加入结晶紫染液，对已迁移RAW264.7细胞染色3 min，吸去染液后加入PBS洗涤3次。用棉签将上室内未迁移细胞擦去，将Transwell培养室放置于光学显微镜下观察并计算4组RAW264.7细胞的迁移数量。

1.2.4. 免疫荧光染色检测细胞骨架

5组细胞以2×104/孔密度接种于6孔板中，培养5 d后，用移液枪将孔板内培养基吸去，加入4%多聚甲醛固定细胞，吸去固定液后加入PBS洗涤3次，加入0.5% Triton X-100(Sigma Aldrich)透化20 min，吸去Triton X-100后PBS洗涤3次，加入FITC标记的phalloidin(1:1000，Sigma Aldrich)，在37 ℃下孵育3 h，PBS洗涤后用碘化丙啶在室温下对细胞核染色5 min，经PBS洗涤晾干用荧光显微镜观察。

1.2.5. TRAP染色检破骨细胞分化

实验增加第5组(高糖+ZOL+SB203580组)：在浓度16.5 mmol/L葡萄糖培养基内加入0.1 μmol ZOL后加入10 μmol p38 MAPK拮抗剂SB203580。5组RAW264.7细胞诱导分化7 d后，将每组细胞的培养基吸去，加入PBS洗涤3次，加入4%多聚甲醛固定20 min，按照TRAP染色试剂盒说明进行染色染，染色后避光孵育2 h，去离子水洗涤后自然晾干并观察。检测标准：光学显微镜100倍下，观察并计数破骨细胞生成数量(细胞核≥3个)。

1.2.6. 骨吸收功能检测

选取临床中新拔除的智牙，用口腔科高速手机将牙齿切割0.5 cm×0.5 cm×1 cm的牙片后将牙釉质打磨去除。将牙磨片放置于24孔板内后接种各组细胞，诱导分化9 d后，加入4%多聚甲醛固定10 min，将各组牙本质磨片先后置于1 mol/L氢氧化铵和蒸馏水中在超声震荡机50 Hz频率下交替洗涤共3次。随后，分别使用2.5%戊二醛和1%饿酸固定细胞，分别固定2 h，用浓度为70%、80%、90%、无水乙醇的梯度乙醇进行脱水，加入醋酸异戊酯置换，干燥后在牙本质磨片表面镀金，置于扫描电子显微镜下观察(HITACHI s-4800)。扫面电子显微镜放大500倍，从5组中随机选取5个视野，应用医学数字图像分析系统Med 6.0计算5个视野中吸收陷窝的平均数量和平均面积。

1.2.7. Real-time PCR检测

收集5组细胞，提取总RNA。使用逆转录试剂盒((ReverTra Ace qPCR RT Master Mix)将5组细胞总RNA逆转录为cDNA。按照SYBR®Green real time PCR Master Mix(TOYOBO)的操作说明进行实时聚合酶链反应，PCR产物用StepOnePlus实时PCR系统(Thermo Fisher)检测。按照95 ℃，1 min; 95 ℃，15 s，60 ℃，15 s; 72 ℃，45 s的反应程序，共进行40个循环; 待实时聚合酶链反应完成后进行熔融曲线分析。实验重复3次。引物序列如下所示：

p38 MAPK-F: 5'-CAAGGTCACTGGAGGAAT-3'，

p38 MAPK-R: 5'-GCACTTCACGATGTTGT-3';

p-p38 MAPK-F: 5'-GGCTGGTTGTAGTGAATG-3'，

p-p38 MAPK-R: 5'-GTCTCTGTCTCCTTCTGTT-3';

NFATc1-F: 5'-CCGAGGAAGAACACTACA-3'，

NFATc1-R: 5'-TGATTGGCTGAAGGAACA-3';

CTSK-F: 5'-TTGTGACCGTGATAATGTG-3'，

CTSK-R: 5'-TTATTCCGAGCCAAGAGA-3';

TRAP-F: 5'-TCGGCTTCTTCTCCAATC-3'，

TRAP-R: 5'-GACCTCCAAGTTCTTATCCT-3';

GAPDH-F: 5'-AGGCCGGTGCTGAGTATGTC-3'，

GAPDH-R: 5'-TGCCTGCTTCACCACCTTGT-3'。

1.2.8. Western blot检测

用裂解液提取5组细胞的总蛋白，冰上孵育后将获得的蛋白保存备用。使用BCA蛋白检测试剂盒(Beyotime)在酶标仪中测定蛋白浓度，加入5倍浓度上样缓冲液，在沸水中反应10 min，凝胶配置好后，凝胶电泳并将蛋白质转移到PVDF膜上; 用封闭液(5%牛血清蛋白)将膜封闭2 h后，用TBST洗涤3次，分别加入一抗：兔抗小鼠p38 MAPK一抗(1:1000，Cell signalling technology)、p-p38 MAPK一抗(1:1000，Cell signalling technology)、NFATc1一抗(1:2000，Abcam)、CTSK一抗(1:500，GeneTex)、TRAP一抗(1:1000，Abcam)、GAPDH一抗(1:2000，Santa Cruz Biotechnology)，4 ℃孵育过夜，将GAPDH作为内参; TBST洗涤3次后加入二抗：羊抗兔二抗(1:5000，KPL)于室温下孵育1 h。将冲洗好的条带扫描成像后，应用Image J软件对膜上蛋白质条带的灰度值进行检测分析。实验重复3次。

1.2.9. 统计学分析

实验中计量资料应用x±s表示，使用SPSS(22.0)统计学软件对实验数据进行分析，应用单因素方差分析对5组数据进行比较，采用Dunnett方法进行两组间差异比较。P < 0.05为差异具有统计学意义。

2. 结果

2.1. ZOL对RAW264.7细胞在高糖条件下细胞增殖活性的影响

对各组RAW264.7细胞培养24、48、72 h后，发现ZOL对细胞增殖活性无显著影响; 应用MTT法测定各组细胞增殖情况，结果显示：在细胞培养24、48、72 h后，4个组别RAW264.7细胞增殖活性未见明显差别(图 1)，差异无统计学意义。

1.

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诱导培养24，48，72 h后，ZOL对高糖条件下RAW264.7细胞活性的影响

Effect of ZOL on viability of RAW264.7 cells cultured in high glucose condition evaluated by MTT assay.

2.2. ZOL对RAW264.7细胞在高糖条件下细胞迁移能力的影响

Transwell培养室对各组细胞在诱导培养后，48 h各组细胞迁移数量高于24 h。与低糖组相比，高糖组RAW264.7细胞迁移数量显著升高，低糖+ZOL组细胞的迁移能力降低; 与高糖组相比，高糖+ZOL组的细胞迁移能力显著下降(图 2)。

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诱导培养24，48 h后，ZOL对高糖条件下RAW264.7细胞迁移的影响

Effects of ZOL on migration of RAW264.7 cells in high glucose condition assessed using Transwell assay. *P < 0.05 vs low group; **P < 0.05 vs low group; ***P < 0.05 vs high group.

2.3. ZOL对RAW264.7细胞在高糖条件下细胞增骨架清晰程度的影响

免疫荧光化学染色实验表明，低糖组细胞骨架最清晰，破骨细胞封闭区的形成更加完整; 高糖条件抑制破骨细胞细胞骨架的形成，对破骨细胞封闭区的形成具有抑制作用; ZOL加入后，高糖+ZOL组的细胞骨架的清晰程度进一步下降，破骨细胞封闭区的完整性受到进一步破坏(图 3)。

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ZOL对高糖条件下各组细胞骨架清晰程度的影响

ZOL inhibits cytoskeleton clarity of the cells cultured in high glucose condition. The cytoskeleton was labeled with FITC-phalloidin and cell nuclei with PI (scale bar=20 μm).

2.4. ZOL对RAW264.7细胞在高糖条件下分化生成的影响

在诱导破骨细胞分化7 d后，各组细胞进行TRAP染色。低糖组分化生成的破骨细胞数高于高糖组和低糖+ZOL组; 与高糖组相比，高糖+ZOL组分化生成的破骨细胞数目减少，高糖+ ZOL+SB203580组分化生成的破骨细胞数目显著减少，高糖+ZOL+SB203580组分化生成的破骨细胞数目显著减少(图 4)。

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ZOL对高糖条件下各组破骨细胞分化生成的影响

ZOL inhibits osteoclast differentiation under high glucose condition. A, B: TRAP staining of osteoclasts after 7 days of culture (× 400). *P < 0.05 vs low group; **P < 0.05 vs low group; ***P < 0.05 vs high group; #P < 0.05 vs high+ ZOL group.

2.5. ZOL对RAW264.7细胞在高糖条件下骨吸收功能的影响

将细胞于牙本质磨片上诱导培养9 d后，通过扫面电子显微镜检测各组破骨细胞骨吸收陷窝的面积和数量。实验结果显示：低糖组骨吸收陷窝数量、面积高于低糖+ZOL组和高糖组，高糖+ZOL组的吸收陷窝数量和面积低于高糖组，高糖+ZOL+SB203580组骨吸收陷窝数、面积低于高糖+ZOL组; 在5个组别中，低糖组的骨吸收陷窝数量最多、面积最大; 高糖+ZOL+SB203580组骨吸收陷窝数量最少、面积最小(图 5)。

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ZOL对高糖条件下各组破骨细胞骨吸收功能的影响

ZOL inhibits bone resorption by osteoclasts under high glucose condition. (A, B) Resorption lacunaes formed on dentin slices were visualized by SEM(×500). *P < 0.05 vs low group; **P < 0.05 vs low group; ***P < 0.05 vs high group; #P < 0.05 vs high+ ZOL group. Each bar is mean±SD of at least three independent experiments.

2.6. 实时荧光定量PCR检测ZOL和葡萄糖水平对p38 MAPK通路相关基因mRNA表达的影响

高糖组p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP mRNA水平均低于低糖组，ZOL加入后，p38MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP mRNA水平进一步降低; 高糖+ZOL组的p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP mRNA水平相比于高糖组降低。加入p38 MAPK拮抗剂SB203580后，高糖+ZOL+SB203580组的p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP mRNA水平与高糖+ZOL组比较均降低(图 6)。

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实时荧光定量PCR检测p38 MAPK、p- p38 MAPK、NFATc1、CTSK、TRAP mRNA表达

Detection of p38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP mRNA expression by real-time PCR. *P < 0.05 vs low group; **P < 0.05 vs low group; ***P < 0.05 vs high group; #P < 0.05 vs high+ZOL group.

2.7. Western blot检测ZOL和葡萄糖水平对p38 MAPK通路相关蛋白表达的影响

与低糖组相比较，高糖组p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP蛋白水平均降低，ZOL的加入后，p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP蛋白水平进一步降低; 高糖+ZOL组的p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP蛋白水平相比于高糖组降低; 加入p38 MAPK拮抗剂SB203580后，高糖+ZOL+SB203580组的p38 MAPK、p-p38 MAPK 、NFATc1、CTSK 和TRAP 蛋白水平与高糖+ZOL组比较均降低(图 7A~E)。

7.

7

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Western blotting检测p38 MAPK、p-p38 MAPK、NFATc1、CTSK、TRAP蛋白表达

Detection of p38 MAPK, p-p38 MAPK, NFATc1, CTSK and TRAP protein expression by Western blotting. *P < 0.05 vs low group; **P < 0.05 vs low group; ***P < 0.05 vs high group; #P < 0.05 vs high+ZOL group.

3. 讨论

本研究证实高糖条件对破骨细胞分化生成及骨吸收功能发挥抑制作用，高糖条件抑制p38 MAPK、p-p38 MAPK的表达，同时抑制NFATc1、CTSK、TRAP等破骨细胞特异性因子的表达; 随着ZOL的加入，破骨细胞分化生成及骨吸收功能受到进一步的抑制，p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP的表达进一步降低。本研究在组间比较中加入p38 MAPK拮抗剂SB203580，结果证实，ZOL通过p38 MAPK通路抑制高糖条件下破骨细胞分化生成和骨吸收，对p38 MAPK信号通路调控的下游破骨细胞相关因子NFATc1、CTSK和TRAP mRNA及蛋白的表达产生影响，从而实现了对高糖条件下破骨细胞分化生成和骨吸收功能的作用。

血糖升高是糖尿病的主要病理表现。目前，有关高糖条件对骨代谢相关细胞破骨细胞影响的研究较为集中[16]。高浓度的葡萄糖减少了破骨细胞的形成，并且抑制了2型糖尿病大鼠破骨细胞的分化和功能[17]。研究证实，2型糖尿病大鼠BMPs和FGF表达降低，骨钙素表达降低，骨结合减少[18]。Maycas[19]发现高糖水平通过阻碍破骨细胞样细胞形成中羟基磷灰石的吸收，抑制RAW264.7细胞的分化。还有研究表明，高葡萄糖条件降低了ATP6v0d2和DC-STAMP基因的表达，从而抑制了破骨细胞前体细胞的融合进而分化为破骨细胞的能力[20]。，在高糖的条件下，RAW264.7细胞具有更强的迁移能力，但破骨细胞的分化生成和骨吸收功能在高糖的条件下受到明显抑制; 低糖组分化生成破骨细胞数量高于高糖组，牙本质切片中低糖组破骨细胞骨吸收陷窝的数量和面积高于高糖组。

研究表明，p38 MAPK通路对破骨细胞分化生成发挥重要作用[21-23]; 阻断p38 MAPK信号通路可抑制RANKL诱导的破骨细胞分化生成[24]。在M-CSF和RANKL诱导的原代骨髓细胞培养中，MKK3或MKK6的缺失可导致p38 MAPK活性降低，NFATc1、CTSK、Oscar和MMP9等破骨细胞标志基因表达减少，骨吸收功能受损[25]。研究发现，通过RANKL-RANK-TRAF6轴激活p38 MAPK，促进了破骨细胞前体的增殖分化，MAPK信号通路可调控RANKL诱导的破骨细胞前体增殖分化[26]。本实验经Real-time PCR和Western blotting检测发现，高糖抑制p38 MAPK和磷酸化p38 MAPK的表达，同时抑制破骨细胞分化生成特异性因子NFATc1、TRAP和骨吸收功能的特异性因子CTSK的表达。随着p38 MAPK通路抑制剂SB203580的加入，TRAP染色检测和骨吸收陷窝检测均证明ZOL通过p38 MAPK通路抑制高糖条件下破骨细胞分化生成及骨吸收功能。

目前，已有相关研究显示双膦酸盐可用于糖尿病性骨质疏松的治疗。在糖尿病早期，应用唑来膦酸可预防骨质疏松[27-28]。双膦酸盐可逆转高糖条件促进骨吸收的过程，改善2型糖尿病骨质疏松患者种植体周围骨整合效果[29]。Lee等[30]通过实验发现，阿仑膦酸盐(ALN)可提高糖尿病骨质疏松症大鼠的骨密度和骨硬度，发挥抵抗骨吸收的作用。临床实验病例证实，应用ALN对老年2型糖尿病患者进行治疗，患者的骨密度相对安慰剂组有所增加[31]。对2型糖尿病模型大鼠皮下注射双膦酸盐类药物利塞膦酸盐(RIS)，研究发现，RIS的应用可减少破骨细胞数量、抑制骨吸收功能，并增加股骨椎体矿物质含量和骨密度[32]。本研究结果显示，ZOL抑制了高糖条件下RAW264.7细胞的迁移能力，并进一步抑制破骨细胞的分化生成和骨吸收功能，该结果可能与p38 MAPK、p-p38 MAPK、NFATc1、CTSK和TRAP的表达受到抑制相关。

综上所述，高糖抑制p38 MAPK、p-p38 MAPK及破骨细胞特异性因子NFATc1、CTSK、TRAP的表达，抑制破骨细胞分化生成及其骨吸收功能；ZOL通过调控p38 MAPK通路抑制高糖条件下破骨细胞分化和骨吸收功能。本实验研究结果为双膦酸盐抗糖尿病性骨质疏松治疗的分子机制及临床应用提供了理论基础并拓展了新思路，提示p38 MAPK有潜力成为治疗糖尿病骨质疏松症的新靶点。

Biography

蔺一凡，在读硕士研究生，E-mail: 765240845@qq.com

Funding Statement

河北省高等学校科学研究计划(QN2020438)；河北省自然科学基金项目(H2017209114)；河北省卫计委重点科技研究计划(20180745)

Contributor Information

蔺 一凡 (Yifan LIN), Email: 765240845@qq.com.

董 伟 (Wei DONG), Email: 970484328@qq.com.

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