趙艷芳+曲寶明+劉文東+馬海燕

[摘要] 目的 研究臍血鉛濃度與新生兒端粒長度的相關(guān)性。 方法 收集2010年7月～2013年4月青島市市立醫(yī)院產(chǎn)科孕期無急慢性疾病的足月順產(chǎn)兒臍血78例，原子吸收光譜法測定臍血鉛濃度，測得臍血鉛濃度在11～89?g/L范圍。將78例臍血分成A、B、C三組：A組臍血鉛濃度<30?g/L，共28例，B組臍血鉛濃度30～60?g/L，共38例，C組臍血鉛濃度>60?g/L，共12例。采用全血提取基因組法提取臍血DNA，熒光定量PCR法測基因組DNA的端粒長度。各組間比較采用單因素方差分析，兩兩比較采用q檢驗(yàn)，相關(guān)性采用Spearman相關(guān)性研究。 結(jié)果 A組端粒長度為：1.152±0.716，B組端粒長度為0.614±0.407，C組端粒長度為0.546±0.339，從A組到C組端粒長度逐漸縮短，且差異有統(tǒng)計學(xué)意義（F=4.895，P<0.05）。B組端粒長度低于A組，差異有統(tǒng)計學(xué)意義（P<0.05），C組端粒長度低于A組端粒長度，差異有統(tǒng)計學(xué)意義（P<0.01）。B、C兩組之間的差異沒有統(tǒng)計學(xué)意義（P>0.05）。三組端粒長度與臍血鉛濃度呈負(fù)相關(guān)（r=-0.461，P<0.01）。 結(jié)論 臍血鉛可導(dǎo)致新生兒端粒長度的縮短，且隨臍血鉛濃度的升高，新生兒端粒長度進(jìn)行性縮短，端粒長度縮短影響細(xì)胞分裂，決定細(xì)胞壽命。

[關(guān)鍵詞] 臍血；鉛；新生兒；端粒長度

[中圖分類號] R743.3 [文獻(xiàn)標(biāo)識碼] A [文章編號] 2095-0616（2014）06-07-05

A correlation study of umbilical cord blood lead concentrations and neonatal telomere length

ZHAO Yanfang QU Baoming LIU Wendong MA Haiyan

Department of Paediatrics，Affiliated Qingdao Municipal Hospital of Qingdao University Medical College，Qingdao 266000，China

[Abstract] Objective To study the correlation of umbilical cord blood lead concentrations and neonatal telomere length. Methods We collected 78 cases of umbilical cord blood from term infants whose gestation was without acute or chronic disease from Qingdao Municipal Hospital between July of 2010 to April of 2013.We used atomic absorption spectrometry to determine the concentration of umbilical cord blood lead. The umbilical cord blood lead concentrations measured range was between 11 and 89 ?g/L. The 78 cases of umbilical cord blood was divided into three groups：A，B，and C.Umbilical cord blood lead concentrations for group A was <30?g/L（n=28）；group B was 30-60 ?g/L （n=38）；and group C was>60 ?g/L （n=12）.To extract umbilical cord blood DNA，extraction genome DNA from whole blood was used. Fluorescence quantitative PCR was used to measure the telomere length of genome DNA.Data were assessed by one-way analysis of variance，differences between groups were compared by q-test and the relationship were analyzed with Spearman correlation research. Results The telomere length of group A was 1.152±0.716，the telomere length of group B was 0.614±0.407，and the telomere length of group C was 0.546±0.339.The telomere length shortened gradually from group A to group C，and the difference was statistically significant（F=4.895，P<0.05）.Telomere length of group B was shorter than group A， the difference was statistically significant （P<0.05），the group C was shorter than group A，the difference was statistically significant （P<0.01）.There was no statistical significance between group B and C （P>0.05）.The relationship of the three group's telomere length and umbilical cord blood lead concentrations correlated negatively（r=-0.461，P<0.01）. Conclusion Umbilical cord blood lead can shorten the neonatal telomere length. With increasing blood lead concentration，the telomere length of the neonate are progressive shorten which influenced cell division and determined cellular age.endprint

[Key words] Umbilical cord blood；Lead；Neonate；Telomere length

Telomere，the outer most ends of the chromosomes，is essential for chromosomal stability and integrity.Telomere

consists of repeats of DNA sequences，and its function is to stabilize chromosome to prevent end-to-end fusion between chromosomes，degradation， rearrangement，and chromosome loss.As the normal somatic cell undergoes mitosis constantly，so telomere length shortens correspondingly. When telomere reaches a critical length，it can prevent the further division of the cell，which causes cell aging and death. Neonatal lead exposure is mainly transferred from the mother during pregnancy through the umbilical cord and placental barrier，Wu Y[1] et al found that long lead exposure can induce telomere length shortening.This study may provide a new theoretical basis for the relationship between blood lead and infant growth and development through understanding the change of telomere length with newborn umbilical cord blood lead concentrations.

1 Materials and methods

1.1 Materials

1.1.1 Main reagent and instrument TE buffer solution and synthetic primer were bought from Sangon Biotech（Shanghai）Co.，Ltd；Blood Genome DNA Extraction Kit and SYBR?Premix Taq? Ⅱ（Perfect Real Time） was purchased from Takara company.The instruments used：Bio-rad fluorescence ration PCR，Ultraviolet spectrophotometer，Hydroextractor，and Electrophoretic apparatus.

1.1.2 Experimental samples and samples processing 78 cases of umbilical cord blood was collected from term infants whose gestation was without acute or chronic disease from Qingdao Municipal Hospital between July of 2010 to April of 2013.And 4ml of umbilical blood was removed from the umbilical vein and placed into two anticoagulant tubes，which were then centrifuge for 10 minutes at 1800r/min.The serum and plasma were placed into EP containing tubes. The 78 cases were divided into three groups： A， B，C with blood lead concentrations ranging between 30 and 60?g/L.Group A umbilical cord blood lead concentrations were<30?g/L，a total of 28 cases； group B 30-60?g/L，a total of 38 cases；group C>60 ?g/L，a total of 12 cases.The blood，serum and plasma were refrigerated together at -80℃.

1.2 Methods

1.2.1 Extract umbilical cord blood DNA and Fluorescence quantitative PCR Used blood genome DNA extraction kit to extract genome from umbilical cord blood and do the operation steps strictly with， ultraviolet spectrophotometer instrument to measure the DNA concentration and purity，and selected the OD260/OD280 between 1.8 and 2.0 as example.The fluorescence quantitative PCR kit for fluorescence quantitative PCR was used to determinate the telomere length，and got Ct values of telomere and 36B4 from PCR.The PCR reaction system was as follows：SYBR?Premix EX Taq? Ⅱ（2X）10μL， Primer （concentration was 10μmol/L）0.8μL，ROX Reverence Dye（50X）0.4μL，DNA templates 2.0ul and ddH2O 6.0μL. The primer sequences of telomeric gene was：Forward primer，5-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3；endprint

Reverse primer，5-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3；primer sequences of 36B4 gene was： Forward primer，5-CAGCAAGTGGGAAGGTGTAATCC-3；Reverse primer，5-CCCATTCTATCATCAACGGGTACAA-3. Cycling conditions （for both telomere and 36B4）were：10min at 95℃，followed by 40 cycles of 95℃，for 5s， 56℃for 30s，and 72℃ for 60s.

1.2.2 Make standard curve and calculate telomere length Select one sample to dilute （dilution factor is five） according to the ratio of equality as the standard preparation， they were： 0.016ug/L，0.08ug/L， 4ug/L，20ug/L，and 100ug/L. Then got Ct value by PCR，and according to Ct value of standard preparation and itsconcentration to make standard curve.Last，reckon the telomere length by the Ct value.The calculation formula of telomere length was：telomere length=2-ΔΔCt，ΔΔCt=ΔCtstandard preparation-ΔCtexample，ΔCtexample=Ct36B4-Cttelomere，ΔCtstandard preparation =Ct36B4-Cttelomere.

1.3 Statistics analysis

SPSS17.0 was used to make one-factor analysis of variance between groups by the relative telomere length and different umbilical cord blood lead concentrations.The differences between groups were compared by q-test.Spearman correlation research can be used to analyze the relationship between umbilical cord blood lead and telomere length.

2 Results

2.1 Standard curve and molten curve

Nonspecific products did not be found in melting curve appears（figure 2）.

Figure 1 Telomere standard curve

Temperature， Celsius

Temperature， Celsius

Figure 2 Molten curve of telomere and 36B4

2.2 Concentration of umbilical cord blood lead

Group A cord blood lead concentrations < 30?g/L，a total of 28 cases；group B 30-60?g/L，a total of 38 cases； group C >60 ?g/L，a total of 12 cases.

2.3 Average telomere length

The telomere length of group A was 1.152±0.716，the telomere length group B was 0.614±0.407，and the telomere length group C was 0.546±0.339.The telomere length shortened gradually from group A to group C and the difference showed statistical significance（F=4.895，P<0.05）.Telomere length of group B was shorter than group A，the difference showed statistical significance（P<0.05），the group C was shorter than group A，the difference showed statistical significance（P<0.01）.Those indicated that the increase of blood lead concentrations could lead to telomere length shorten，and when the umbilical cord blood lead concentrations exceeded 30 ?g/L，neonatal telomere length was affected severely.endprint

2.4 The relationship of telomere length and blood lead concentration

The relationship between telomere length and blood lead concentration was a negative correlation （r=-0.461， P<0.01），and with increasing blood lead concentration，the telomere length of neonate were progressive shorten.

3 Conclusions

3.1 The relationship between telomere length and diseases

Telomere length has been associated with many systemic diseases. A Study found that[2]there was a close relationship between the change of telomere length and coronary heart disease，as telomere length shortens increases the incidence of cardiovascular disease.It is possible that telomere length shortening is an important risk factor for atherosclerosis，which leads to the higher incidence of coronary heart disease[3].Another research found that[4] telomere length shorten increased the total incidence and mortality of COPD. Many clinical studies showed that the abnormal of average telomere length of peripheral blood was significantly associated with the increased cancer risk.Existing studies have shown that[5] telomere dysfunction caused by telomere shorten was the reason of a tumor early chromosome fusion；in the late tumor，telomere shorten further intensified fracture fusion and internal double-stranded DNA genomic instability.Telomere was also associated with the occurrence of a variety of autoimmune diseases，such as Systemic Lupus Erythematosus， Rheumatoid Arthritis， Diabetes， Hyperthyroidism and other diseases. Research showed[6] shorten telomere length could increase the risk of Myelodysplastic Syndrome，and may be also be one of the reasons of hypertension. For children，the shorten telomere length could affect the development of the nervous system，and lead to mental development disorder[7].

Telomere is composed of telomere DNA and telomere protein and telomere length is regarded as a marker for cell biological age[8].Studies have shown that[9] telomere length is cut 4.8% of its length per decade. In normal human somatic cells，telomere progressively shortens with the cell mitosis.When the telomere reaches a critical length，it can prevent the further division of the cell，causing cell aging and death[10].But there is a big difference between the length of the telomere of different individuals of the same age.Heredity accounts for 40-80% of these differences[11]. Of the genetic factors，telomere length is mainly determined by the regulation of telomerase[12]，and telomere binding protein also play an important role in the maintaining of telomere length.Telomerase primarily includes telomerase RNA（hTERC） and telomerase catalytic subunit（hTERT）[13-14].If telomerase is lacking，telomere progressively shortens with the cell mitosis， and if the telomeres shorten to a certain length，cell senescence and apoptosis happened[15].Telomere binding protein consisted of 6 parts[16-17]：TRF1，TRF2， TIN2，TPP1，POT1 and Rap1.Telomere binding protein can guide the telomerase with associated chromosomes parts and protect the chromosomes. Therefore， telomere length can be maintained by the telomerase，the telomere binding proteins， and the interactions of both.endprint

In addition to genetic factors，telomere length also be affected by environment.Of the environmental factors， lead was the biggest threat to neonatal growth. Lead enters the body through the respiratory tract and is not easily expelled. It can cause a variety of viscera damage to human body.It can affect telomere length extension，cell survival， and interfere with cell function. A study found that long lead exposure can induce telomere length shortening. Some studies also showed that[18]in the lead exposure environment，telomerase activity decreases could influence the regulation of telomere length. Therefore， the change of telomere length in the neonate may be associated with lead exposure.

3.2 The effect and meaning of blood lead concentration of cord blood to telomere length of neonatus

At present，the concentration of lead poisoning in our country is defined as blood lead concentrations >100?g/L.Lead does not have any normal physiological functions in the human body，but lead can affect a variety of system functions and is not easily removed after it enters the body. Without any normal physiological functions to in human body，its ideal blood lead concentrations should be 0. As a result， some developed countries lowered the lower limit defining concentration for lead poisoning，setting the blood lead concentrations <30?g/L as safe，and >60?g/L as lead poisoning. For the fetus，lead is mainly transferred by the mother during pregnancy through the umbilical cord and placental barrier.Therefore，this study collected umbilical cord blood and different of umbilical cord blood lead concentrations were divided into three groups： group A cord blood lead concentrations <30?g/L， group B 30-60?g/L， and group C >60?g/L. This allowed us to study the effect of umbilical cord blood lead to neonatal telomere length.From these result we determined that with the rise of umbilical cord blood lead concentration，the telomere length shorten progressively，and telomere length and umbilical cord blood lead concentrations correlated negatively（r=-0.461，P<0.01）. Umbilical cord blood lead could cause neonatal shortening of telomere length； moreover，different umbilical cord blood lead concentration had different influence to telomere length.The telomere length of group A was 1.152±0.716，the telomere length of group B was 0.614±0.407，and the telomere length of group C was 0.546±0.339.The difference between these groups were statistically significant （P<0.05），that is to say，the effect of telomere length was different with varying neonatal umbilical cord blood lead concentrations.Among them，the average telomere length of group B was shorter than group A，the difference was statistically significant（P<0.05），the group C was shorter than group A，the difference was statistically significant（P<0.01）.This data suggests that when the blood lead concentrations > 30 ?g/L， telomere length would seriously be affected and cause telomere shortening.The latest study suggested that the two main factors[19]for telomere shortening were the incomplete copy of cell division telomere end and the damage of telomere DNA. Under normal condition， these two factors could be recover telomere length under the action of telomere and the binding of telomerase and telomere protein. But when there was lead exposure， telomere shortening could not be restored， telomerase and telomere binding protein function was affected by lead and caused the telomere length shortening. Accordingly we conclude，umbilical cord blood lead can shorten the neonatal telomere length；with increasing blood lead concentration，the telomere length of neonate are progressively shortened；and lead may be interfere with telomere length and make it short by influencing the role of telomerase and telomere binding protein.endprint

In summary，umbilical cord blood lead can shorten the length of neonatal telomere and different neonatal umbilical cord blood lead concentrations are associated with different changes to telomere length. When umbilical cord blood lead exceeds 30?g/L，the newborn telomere length will be affected seriously， and the higher the concentration，the more serious the impact on neonatal telomere length. The length of the telomere shortening can also increase the incidence of a number of diseases，such as Coronary Heart Disease，Hypertension，Cancer，Diabetes，Obstructive Sleep Apnea Syndrome， and a variety of autoimmune diseases.It also can affect children's mental development.Therefore，we should pay more attention to the influence of maternal lead exposure during pregnancy on neonatal growth and development.The influence of blood lead on telomere length may be by affected telomerase and telomere binding protein function that makes the telomere length shortened， however the exact mechanism remains to be further studied.

Reference

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[9] Shiels PG，McGlynn LM，Maclntyre A，et al.Accelerated Telomere Attrition is Associated With Relative Household Income，Diet and Inflammation in the pSoBid Cohort[J].PLoS One，2011，6（7）：e22521.endprint

[10] Entringer S，Epel ES，Kumsta R，et al.Stress exposure in intrauterine life is associated with shorter telomere length in young adulthood[J].Proc Natl Acad Sci USA，2011，108（33）：E513-E518.

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[12] Jeon HS，Choi JE，Jung DK，et al.Telomerase activity and the risk of Lung Cancer[J].J Korean Med Sci，2012，27（2）：141-145.

[13] Liu H，Liu S，Wang H，et al.Genomic amplification of the human telomerase gene（hTERC） associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer[J].Diagn Pathol，2012，7：147-154.

[14] Blackburn EH，Greider CW，Szostak JW.Telomeres and telomerease： the path from maize， Tetrahymena and yeast to human cancer and aging[J].Nat Med，2006，12（10）：1133-1138.

[15] Pan J，Zhang S.Interaction between cationic zinc porphyrin and lead ion induced telomeric guanine quadruplexes：evidence for end-stacking[J].J Biol Inorg Chem，2009，14（3）：401-407.

[16] Qing YF，Zhou JG，Xing Y，et al.Expression of TPP1 and POT1 mRNA in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus[J].Chin J Rheumatol，2010，14（1）：56-59.

[17] de Lange. T Shelterin： the protein complex that shapos and safeguards human telomeres[J].Genes Dev， 2005，19（18）： 2100-2110.

[18] Cui QH，Tang CC，Huang YG.Effects of lead and selenium on telomere binding protein Rap1p，telomerase and telomeric DNA in Saccharomyces cerevisiae[J].Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao （Shanghai），2002，34（2）：240-244.

[19] Bin B，Leng SG，Cheng J，et al.Association between telomere and aromatic hydroearbom. Chin J Prev Med，2010，44（6）：535-538.

（收稿日期：2014-01-26）endprint

[10] Entringer S，Epel ES，Kumsta R，et al.Stress exposure in intrauterine life is associated with shorter telomere length in young adulthood[J].Proc Natl Acad Sci USA，2011，108（33）：E513-E518.

[11] Zhu H，Belcher M，van der Harst P.Healthy aging and disease： role for telomere biology？[J]Clin Sci（Lond），2011，120（10）：427-440.

[12] Jeon HS，Choi JE，Jung DK，et al.Telomerase activity and the risk of Lung Cancer[J].J Korean Med Sci，2012，27（2）：141-145.

[13] Liu H，Liu S，Wang H，et al.Genomic amplification of the human telomerase gene（hTERC） associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer[J].Diagn Pathol，2012，7：147-154.

[14] Blackburn EH，Greider CW，Szostak JW.Telomeres and telomerease： the path from maize， Tetrahymena and yeast to human cancer and aging[J].Nat Med，2006，12（10）：1133-1138.

[15] Pan J，Zhang S.Interaction between cationic zinc porphyrin and lead ion induced telomeric guanine quadruplexes：evidence for end-stacking[J].J Biol Inorg Chem，2009，14（3）：401-407.

[16] Qing YF，Zhou JG，Xing Y，et al.Expression of TPP1 and POT1 mRNA in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus[J].Chin J Rheumatol，2010，14（1）：56-59.

[17] de Lange. T Shelterin： the protein complex that shapos and safeguards human telomeres[J].Genes Dev， 2005，19（18）： 2100-2110.

[18] Cui QH，Tang CC，Huang YG.Effects of lead and selenium on telomere binding protein Rap1p，telomerase and telomeric DNA in Saccharomyces cerevisiae[J].Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao （Shanghai），2002，34（2）：240-244.

[19] Bin B，Leng SG，Cheng J，et al.Association between telomere and aromatic hydroearbom. Chin J Prev Med，2010，44（6）：535-538.

（收稿日期：2014-01-26）endprint

[10] Entringer S，Epel ES，Kumsta R，et al.Stress exposure in intrauterine life is associated with shorter telomere length in young adulthood[J].Proc Natl Acad Sci USA，2011，108（33）：E513-E518.

[11] Zhu H，Belcher M，van der Harst P.Healthy aging and disease： role for telomere biology？[J]Clin Sci（Lond），2011，120（10）：427-440.

[12] Jeon HS，Choi JE，Jung DK，et al.Telomerase activity and the risk of Lung Cancer[J].J Korean Med Sci，2012，27（2）：141-145.

[13] Liu H，Liu S，Wang H，et al.Genomic amplification of the human telomerase gene（hTERC） associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer[J].Diagn Pathol，2012，7：147-154.

[14] Blackburn EH，Greider CW，Szostak JW.Telomeres and telomerease： the path from maize， Tetrahymena and yeast to human cancer and aging[J].Nat Med，2006，12（10）：1133-1138.

[15] Pan J，Zhang S.Interaction between cationic zinc porphyrin and lead ion induced telomeric guanine quadruplexes：evidence for end-stacking[J].J Biol Inorg Chem，2009，14（3）：401-407.

[16] Qing YF，Zhou JG，Xing Y，et al.Expression of TPP1 and POT1 mRNA in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus[J].Chin J Rheumatol，2010，14（1）：56-59.

[17] de Lange. T Shelterin： the protein complex that shapos and safeguards human telomeres[J].Genes Dev， 2005，19（18）： 2100-2110.

[18] Cui QH，Tang CC，Huang YG.Effects of lead and selenium on telomere binding protein Rap1p，telomerase and telomeric DNA in Saccharomyces cerevisiae[J].Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao （Shanghai），2002，34（2）：240-244.

[19] Bin B，Leng SG，Cheng J，et al.Association between telomere and aromatic hydroearbom. Chin J Prev Med，2010，44（6）：535-538.

（收稿日期：2014-01-26）endprint