严重烧伤大鼠骨形成与骨吸收相关指标的变化

龚翔; 叶子青; 余刚; 张伟; 张卫东; 周雪情; 李敏; 谢卫国

doi:10.3760/cma.j.cn501120-20200505-00253

严重烧伤大鼠骨形成与骨吸收相关指标的变化

doi: 10.3760/cma.j.cn501120-20200505-00253

武汉大学同仁医院暨武汉市第三医院烧伤科　　430060

基金项目:

武汉市卫生和计划生育委员会科研项目 WX17Q11

详细信息

通讯作者:
谢卫国，Email：wgxie@hotmail.com

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出版历程
- 收稿日期: 2020-05-05

Changes in the related indicators of bone formation and bone resorption in severely burned rats

Institute of Burns, Tongren Hospital of Wuhan University ＆ Wuhan Third Hospital, Wuhan 430060, China

Funds:

Scientific Research Project of Health and Family Planning Commission of Wuhan City of China WX17Q11

More Information

Corresponding author: Xie Weiguo, Email: wgxie@hotmail.com

摘要

摘要: 目的研究严重烧伤大鼠骨形成及骨吸收相关指标的变化。方法采用实验研究方法。将30只6~8周龄SD雌性大鼠按随机数字表法分为假伤组、12%体表总面积（TBSA）Ⅲ度烧伤组、24%TBSAⅢ度烧伤组，每组10只。于各组大鼠背部进行相应处理，之后仅烧伤大鼠按Parkland公式经腹腔注射补液，创面外涂20 g/L碘伏，直至创面愈合。伤后28 d，取各组大鼠胫骨组织，分别行Masson、抗酒石酸酸性磷酸酶染色，观察新生骨组织、破骨细胞数量；取各组大鼠腹主动脉血制备血清，分别采用甲基百里酚蓝比色法、磷钼酸法测定骨代谢指标血清钙离子、磷离子浓度，采用酶联免疫吸附测定法检测血清中骨形成标志物Ⅰ型前胶原氨基端前肽（P1NP）和骨吸收标志物β-Ⅰ型胶原羧基端肽（β-CTX）水平；取各组大鼠第1腰椎组织，采用实时荧光定量反转录PCR法检测护骨因子、核因子κB受体激活蛋白配体（RANKL）、肿瘤坏死因子受体相关因子6（TRAF-6）、活化T细胞核因子1（NFATC1）、c-Fos、c-Src的mRNA表达水平。数据处理采用单因素方差分析、Bonferroni法、Welch检验、Games-Howell检验、Kruskal-Wallis检验、Mann-Whitney U检验及Bonferroni校正。结果伤后28 d，与假伤组比较，2个烧伤组大鼠胫骨组织中新生骨组织生成均减少，烧伤面积越大减少越明显；2个烧伤组大鼠胫骨组织中破骨细胞数量相近，均较假伤组明显增多。伤后28 d，3组大鼠血清钙离子浓度、血清中β-CTX水平相近（P>0.05）；24%TBSAⅢ度烧伤组大鼠血清磷离子浓度明显高于12%TBSAⅢ度烧伤组（P<0.05），且2个烧伤组大鼠血清磷离子浓度均显著高于假伤组（P<0.01）；24%TBSAⅢ度烧伤组大鼠血清中P1NP水平明显低于假伤组（P<0.01）。伤后28 d，假伤组、12%TBSAⅢ度烧伤组、24%TBSAⅢ度烧伤组大鼠第1腰椎组织中护骨因子mRNA表达水平分别为1.01±0.20、1.71±0.83、2.24±0.51，其中24%TBSAⅢ度烧伤组明显高于假伤组（P<0.01）；24%TBSAⅢ度烧伤组大鼠第1腰椎组织中RANKL mRNA表达水平为1.31±0.17，明显高于假伤组的1.00±0.14与12%TBSAⅢ度烧伤组的0.97±0.10（P<0.01）；12%TBSAⅢ度烧伤组、24%TBSAⅢ度烧伤组大鼠第1腰椎组织中TRAF-6、NFATC1（Z=3.141、3.782）、c-Src mRNA表达水平及12%TBSAⅢ度烧伤组大鼠第1腰椎组织中c-Fos mRNA表达水平均明显高于假伤组（P<0.05或P<0.01）；12%TBSAⅢ度烧伤组大鼠第1腰椎组织中c-Fos、c-Src mRNA表达水平均明显高于24%TBSAⅢ度烧伤组（P<0.01）。结论严重烧伤可引起大鼠新生骨组织生成减少及破骨细胞数量增多，血清磷离子浓度升高与血清中P1NP水平下降，骨组织中护骨因子、RANKL、TRAF-6、NFATC1、c-Fos、c-Src呈升高趋势，且NFATC1、c-Fos、c-Src随烧伤总面积增加呈下降趋势。
- 烧伤 /
- 骨生成 /
- 代谢 /
- 骨转换 /
- 护骨因子/核因子κB受体激活蛋白配体系统
Abstract: Objective To observe the changes in the related indicators of bone formation and bone resorption in severely burned rats. Methods The experimental research method was adopted. Thirty female Sprague-Dawley rats aged 6 to 8 weeks were divided into sham injury group, 12% total body surface area (TBSA) full-thickness burn group, and 24%TBSA full-thickness burn group according to the random number table, with 10 rats in each group. The rats were treated on the back correspondingly, after which, the burned rats were rehydrated by intraperitoneal injection according to the Parkland formula, and the wound was coated with 20 g/L iodophor until wound healing. On post injury day (PID) 28, the tibia tissue of rats in each group was collected. The new bone tissue and the number of osteoclasts were observed after staining with Masson and tartrate-resistant acid phosphatase, respectively. The abdominal aortic blood of rats in each group was harvested for serum preparation. The bone metabolism indexes of serum calcium ion and phosphorus ion concentration were determined by the methyl thymol blue colorimetric method and phosphomolybdic acid method, respectively. The serum levels of bone formation marker of aminoterminal propeptide of type 1 procollagen (P1NP) and bone resorption marker of beta-carboxy-terminated peptide of type Ⅰ collagen (β-CTX) were determined by enzyme-linked immunosorbent assay. The first lumbar spine tissue of rats in each group was collected, and the mRNA expression levels of osteoprotegerin, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor receptor-associated factor 6 (TRAF-6), nuclear factor of activated T cell 1 (NFATC1), c-Fos, and c-Src were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with one-way analysis of variance, Bonferroni method, Welch test, Games-Howell test, Kruskal-Wallis test, Mann-Whitney U test, and Bonferroni correction. Results On PID 28, compared with that in sham injury group, the formation of new bone tissue in the tibia tissue of rats in the two burn groups was decreased, and the larger the burn area, the more obvious the decrease. The numbers of osteoclasts in the tibia tissue of rats in the two burn groups were similar, both significantly more than the number in sham injury group. On PID 28, the serum calcium ion concentration and serum level of β-CTX of rats in the three groups were similar (P>0.05). The serum phosphorus ion concentration of rats in 24%TBSA full-thickness burn group was significantly higher than that in 12%TBSA full-thickness burn group (P<0.05), and the serum phosphorus ion concentrations in the two burn groups were significantly higher than the concentration in sham injury group (P<0.01). The serum level of P1NP of rats in 24%TBSA full-thickness burn group was significantly lower than that in sham injury group (P<0.01). On PID 28, the mRNA expression levels of osteoprotegerin in the first lumbar spine tissue of rats in sham injury group, 12%TBSA full-thickness burn group, and 24%TBSA full-thickness burn group were 1.01±0.20, 1.71±0.83, and 2.24±0.51, respectively, and that in 24%TBSA full-thickness burn group was significantly higher than that in sham injury group (P<0.01). The mRNA expression level of RANKL in the first lumbar spine tissue of rats in 24%TBSA full-thickness burn group was 1.31±0.17, which was significantly higher than 1.00±0.14 in sham injury group and 0.97±0.10 in 12%TBSA full-thickness burn group (P<0.01). The mRNA expression levels of TRAF-6, NFATC1 (Z=3.141, 3.782), and c-Src in the first lumbar tissue of rats in 12%TBSA full-thickness burn group and 24%TBSA full-thickness burn group and the mRNA expression level of c-Fos in the first lumbar tissue of rats in 12%TBSA full-thickness burn group were significantly higher than those in sham injury group (P<0.05 or P<0.01). The mRNA expression levels of c-Fos and c-Src in the first lumbar spine tissue of rats in 12%TBSA full-thickness burn group were significantly higher than those in 24%TBSA full-thickness burn group (P<0.01). Conclusions Severe burns can cause a decrease in the generation of new bone tissue, an increase in the number of osteoclasts and the serum phosphorus ion concentration, and a decrease in the serum level of P1NP in rats. The level of osteoprotegerin, RANKL, TRAF-6, NFATC1, c-Fos, and c-Src in bone tissue showed an increasing trend while the level of NFATC1, c-Fos, and c-Src showed a decreasing trend with the increase of burn area.
- Burns /
- Osteogenesis /
- Metabolism /
- Bone transformation /
- Osteoprotegerin/receptor activator of nuclear factor-κB ligand system
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1 假伤组与2种面积Ⅲ度烧伤组大鼠伤后28 d胫骨组织成骨情况（红色）比较 Masson×100。1A.假伤组新生骨组织覆盖范围广泛；1B.12%体表总面积（TBSA）Ⅲ度烧伤组新生骨组织较图1A少；1C.24%TBSAⅢ度烧伤组新生骨组织较图1A、1B少

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2 假伤组与2种面积Ⅲ度烧伤组大鼠伤后28 d胫骨组织中破骨细胞（→）数量比较抗酒石酸酸性磷酸酶×100。2A.假伤组破骨细胞数量正常；2B.12%体表总面积（TBSA）Ⅲ度烧伤组破骨细胞数量较图2A增多；2C.24%TBSAⅢ度烧伤组破骨细胞数量较图2A增多，与图2B相近

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表1 实时荧光定量反转录PCR法检测大鼠第1腰椎组织中护骨因子/RANKL系统指标的引物序列及产物大小

指标名称	引物序列（5'→3'）	产物大小（bp）
β肌动蛋白	上游：CGTTGACATCCGTAAAGACCTC	110
β肌动蛋白	下游：TAGGAGCCAGGGCAGTAATCT	110
护骨因子	上游：TGTTCTGGTGGACAGTTTGCC	279
护骨因子	下游：CTCTCAATCTCGTCTGGGCTG	279
RANKL	上游：CCATCGGGTTCCCATAAAGTC	148
RANKL	下游：GATGCCTGAAGCAAATGTTGG	148
TRAF-6	上游：AAGCTGTCCTCTGGCAAATATC	193
TRAF-6	下游：CATGTGCAACTGGGTGTTCTC	193
NFATC1	上游：CAGCCACGAGGCTATCCTGT	151
NFATC1	下游：TCTCCCGATGTCTGTCTCCC	151
c-Fos	上游：GAGAAACGGAGAATCCGAAGG	259
c-Fos	下游：TCAAGTCCAGGGAGGTCACAG	259
c-Src	上游：GCAAGATCACTAGACGGGAATC	126
c-Src	下游：TGTCAAAGTCGGATACAGAGAGG	126
注：RANKL为核因子κB受体激活蛋白配体，TRAF-6为肿瘤坏死因子受体相关因子6，NFATC1为活化T细胞核因子1

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表2 假伤组与2种面积Ⅲ度烧伤组大鼠伤后28 d血清骨代谢指标水平比较（x¯±s）

组别	鼠数（只）	钙离子（mmol/L）	磷离子（mmol/L）	P1NP（ng/L）	β-CTX（ng/L）
假伤组	10	2.29±0.28	1.13±0.20	175±42	20±5
12%TBSAⅢ度烧伤组	10	2.29±0.48	1.66±0.21	158±51	21±7
24%TBSAⅢ度烧伤组	10	2.49±0.30	2.02±0.38	121±24	20±5
F值		1.039	26.572	6.790	0.101
P值		0.367	<0.001	0.007	0.904
P₁值		>0.999	0.001	0.684	>0.999
P₂值		0.645	<0.001	0.009	>0.999
P₃值		0.692	0.019	0.137	>0.999
注：TBSA为体表总面积，P1NP为Ⅰ型前胶原氨基端前肽，β-CTX为β-Ⅰ型胶原羧基端肽；F值、P值为组间各指标总体比较所得；P₁值、P₂值分别为12%TBSAⅢ度烧伤组、24%TBSAⅢ度烧伤组与假伤组各指标比较所得；P₃值为12%TBSAⅢ度烧伤组与24%TBSAⅢ度烧伤组各指标比较所得

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表3 假伤组与2种面积Ⅲ度烧伤组大鼠伤后28 d第1腰椎组织中护骨因子/RANKL系统指标mRNA表达水平比较

组别	鼠数（只）	护骨因子（x¯±s）	RANKL（x¯±s）	TRAF-6（x¯±s）	NFATC1［M（P₂₅，P₇₅）］	c-Fos（x¯±s）	c-Src（x¯±s）
假伤组	10	1.01±0.20	1.00±0.14	1.00±0.29	0.95（0.82，1.17）	1.00±0.19	1.01±0.16
12%TBSAⅢ度烧伤组	10	1.71±0.83	0.97±0.10	2.00±1.00	3.01（1.24，3.38）	1.59±0.23	4.60±1.08
24%TBSAⅢ度烧伤组	10	2.24±0.51	1.31±0.17	3.36±1.44	2.07（1.83，2.27）	1.11±0.11	1.84±0.33
统计量值		F=26.051	F=18.267	F=15.966	χ²=16.777	F=29.391	F=71.462
P值		<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
P₁值		0.065	>0.999	0.029	0.006	<0.001	<0.001
P₂值		<0.001	<0.001	0.001	<0.001	0.593	<0.001
P₃值		0.228	<0.001	0.063	0.453	<0.001	<0.001
注：RANKL为核因子κB受体激活蛋白配体，TBSA为体表总面积，TRAF-6为肿瘤坏死因子受体相关因子6，NFATC1为活化T细胞核因子1；统计量值、P值为组间各指标总体比较所得；P₁值、P₂值分别为12%TBSAⅢ度烧伤组、24%TBSAⅢ度烧伤组与假伤组各指标比较所得；P₃值为12%TBSAⅢ度烧伤组与24%TBSAⅢ度烧伤组各指标比较所得

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