Effect of recombinant human growth hormone on gut microbiota of short children based on 16S rDNA sequencing
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摘要:目的
通过16S rDNA高通量测序分析重组人生长激素(rhGH)治疗前后矮身材儿童肠道菌群的变化。
方法选取初次使用rhGH治疗的3~14岁矮身材儿童作为研究对象，按照rhGH使用剂型的不同分为长效组和短效组，每组16例。长效组使用聚乙二醇重组人生长激素(PEG-rhGH)注射液，短效组使用短效rhGH注射液。分别留取2组患儿治疗前和治疗3、6个月时的新鲜粪便标本共83份，采用16S rDNA高通量测序技术结合生物信息分析技术比较2组患儿不同时点肠道菌群组成、丰度的差异及其与生长指标[胰岛素样生长因子-1(IGF-1)、身高标准差积分(SDS)]的相关性。
结果治疗前及治疗3、6个月时, 2组Chao1指数、Pielou e指数依次升高，提示肠道菌群的丰富度、均匀度持续改善; 长效组治疗3个月时Chao1指数高于短效组，差异有统计学意义(P < 0.05)。置换多元方差分析(Per MANOVA)显示F=3.425、P=0.001, 非度量多维尺度(NMDS)分析显示Stress值为0.17, 提示rhGH可在一定程度上调节矮身材儿童肠道菌群组成和结构。随着rhGH使用时间的延长, 2组拟杆菌门、拟杆菌属的丰度和拟杆菌门与厚壁菌门比值(B/F)均逐渐上升，其余优势菌群的丰度呈现波动性变化，差异无统计学意义(P>0.05)。相关性分析显示，长效组治疗6个月时IGF-1、身高SDS均分别与拟杆菌门、拟杆菌属、B/F呈正相关，短效组治疗6个月时IGF-1与拟杆菌门、拟杆菌属、B/F均呈正相关，短效组治疗3个月时IGF-1、身高SDS与双歧杆菌属呈正相关。功能预测结果显示，肠道菌群主要参与碳水化合物代谢和氨基酸代谢等途径。
结论rhGH能够改善矮身材儿童肠道菌群的组成和丰度，短期内PEG-rhGH在改善肠道菌群丰富度方面相较于短效rhGH具有一定优势, rhGH能增加有益菌丰度而发挥协同促生长作用。
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关键词:
- 16S rDNA测序 /
- 重组人生长激素 /
- 矮身材 /
- 儿童 /
- 肠道菌群 /
- 丰度 /
- 胰岛素样生长因子-1
Abstract:ObjectiveTo analyze the changes of gut microbiota of short children before and after recombinant human growth hormone (rhGH) treatment by 16S rDNA high-throughput sequencing.
MethodsShort children aged 3 to 14 years who were first treated with rhGH were selected as study objects, and were divided into long-acting group(n=16) and short-acting group (n=16) according to the dosage form of rhGH, and the long-acting group used polyethylen Glycol Recombinant Human Growth Hormone (PEG-rhGH). In the short-acting group, short-acting rhGH was used. A total of 83 fresh fecal specimens were collected from the two groups before treatment and 3 and 6 months after treatment, respectively. 16S rDNA high-throughput sequencing technology combined with bioinformation analysis technology were used to compare the differences in intestinal flora composition and abundance and explore their correlations with growth indicators[insulin-like growth factor-1 (IGF-1) and height standard deviation score (SDS)]between the two groups at different time points.
ResultsThe Chao1 and Pielou e indexes gradually increased in two groups before treatment, 3 months and 6 months after treatment, indicating consistently improvement of the richness and homogeneity of the gut microbiota. Chao1 index of the long-acting group was higher than that of the short-acting group at 3 months (P < 0.05). Per MANOVA unweighted-unifrac result showed F value was 3.425 and P value was 0.001, and Non-metric Multidimensional Scale (NMDS) analysis showed that Stress value was 0.17, suggesting that rhGH can regulate intestinal flora composition and structure in short children to a certain extent. With the extension of the use time of rhGH, the abundance of Bacteroidota and Bacteroides increased gradually in both groups, and the ratio of Bacteroidota/Firmicutes (B/F) increased gradually, while the abundance of the rest of the flora showed fluctuating changes, but the differences were not statistically significant (P>0.05). Correlation analysis showed that IGF-1 and height SDS were positively correlated with Bacteroidetes, Bacteroidetes and B/F in the long-acting group at 6 months; IGF-1 was positively correlated with Bacteroidetes, Bacteroidetes and B/F in the short-acting group at 6 months; IGF-1 and height SDS were positively correlated with Bifidobacterium in the short-acting group at 3 months. The functional prediction analysis showed that the gut microbiota of each group was mainly involved in carbohydrate metabolism and amino acid metabolism.
ConclusionThe rhGH can improve the composition and abundance of gut microbiota in short children, and PEG-rhGH has an advantage in improving the abundance of gut microbiota in the short term compared with short-acting rhGH, and rhGH plays a synergistic role in growth promotion by increasing the abundance of beneficial bacteria.
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图 1 NMDS分析结果
S0: 短效组治疗前样本; S3: 短效组治疗3个月时样本; S6: 短效组治疗6个月时样本; L0: 长效组治疗前样本; L3: 长效组治疗3个月时样本; L6: 长效组治疗6个月时样本。
表 1 短效组和长效组患儿不同时点身高SDS、IGF-1水平比较(x ± s)
组别 身高SDS IGF-1/(ng/mL) 治疗前 治疗3个月 治疗6个月 治疗前 治疗3个月 治疗6个月 短效组(n=16) -2.16±0.12 -1.78±0.12* -1.45±0.12*# 112.24±54.32 213.22±80.97* 352.74±85.60*# 长效组(n=16) -2.18±0.10 -1.84±0.11* -1.27±0.09*#△ 111.82±44.17 174.01±52.76*△ 331.55±121.87*# 治疗前和治疗3、6个月时，短效组例数分别为16、16、13例，长效组分别为16、12、10例。SDS: 标准差积分; IGF-1: 胰岛素样生长因子-1。与治疗前比较, *P<0.05; 与治疗3个月时比较, #P<0.05; 与短效组比较, △P<0.05。 
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表 2 短效组和长效组不同时点Chao1指数、Shannon指数比较(x ± s)
组别 Chao1指数 Shannon指数 治疗前 治疗3个月 治疗6个月 治疗前 治疗3个月 治疗6个月 短效组(n=16) 253.18±71.87 275.44±55.13 280.07±79.05 5.51±0.47 5.74±0.70 5.62±0.68 长效组(n=16) 310.29±92.48 324.09±82.36* 337.89±87.08 5.85±0.50 5.95±0.42 6.11±0.45 治疗前和治疗3、6个月时，短效组例数分别为16、16、13例，长效组分别为16、12、10例。与短效组比较, *P<0.05。
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表 3 短效组和长效组不同时点Pielou e指数比较(x ± s)
组别 治疗前 治疗3个月 治疗6个月 短效组(n=16) 0.69±0.05 0.70±0.06 0.71±0.04 长效组(n=16) 0.71±0.04 0.72±0.04 0.73±0.03 治疗前和治疗3、6个月时，短效组例数分别为16、16、13例，长效组分别为16、12、10例。
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表 4 不同时点样本的α多样性指数比较(x ± s)
时点 样本数 Chao1指数 Shannon指数 Pielou e指数 治疗前 32 292.86±77.00 5.68±0.51 0.70±0.05 治疗3个月 28 283.57±83.12 5.83±0.60 0.71±0.50 治疗6个月 23 305.21±85.85 5.92±0.49 0.72±0.04 各时点样本数为短效组与长效组的合计样本数。
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表 5 门水平优势菌群的丰度占比和B/F比较
组别 时点 样本数 厚壁菌门/% 拟杆菌门/% 放线菌门/% 变形菌门/% B/F 长效组 治疗前 16 70.76 10.63 11.57 6.45 0.15 治疗3个月 12 62.60 14.88 14.04 4.20 0.24 治疗6个月 10 66.87 18.60 8.31* 4.95 0.28 短效组 治疗前 16 68.95 10.41 15.96 2.19 0.15 治疗3个月 16 64.18 14.45 15.46 4.80 0.23 治疗6个月 13 66.23 16.27 12.96 3.95 0.25 B/F: 拟杆菌门与厚壁菌门比值。与治疗3个月时比较, *P<0.05。
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表 6 属水平优势菌群的丰度占比
% 组别 时点 样本数 布劳特氏菌属 双歧杆菌属 粪杆菌属 拟杆菌属 长效组 治疗前 16 17.15 8.31 13.34 7.95 治疗3个月 12 13.88 10.84 10.41 9.55 治疗6个月 10 16.29 5.40*# 9.93 13.83 短效组 治疗前 16 15.49 13.31 8.73 8.51 治疗3个月 16 15.76 13.43 12.21 11.50 治疗6个月 13 16.67 11.89 12.93 11.79 与治疗3个月时比较, *P<0.05; 与短效组比较, #P<0.05。
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