Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expressi...Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.Methods We cultured MCF-7 human breast cancer cells and established nude mice bearing xenograffs of these cells,and randomly divided them into experimental and control groups.The experimental group was treated with human leptin,while the control group was treated with the same volume of normal saline.A real-time reverse transcriptase-polymerase chain reaction （RT-PCR） assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues.Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells.Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.Results Leptin activated HSP70 in a dose-dependent manner in vitro：leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells （P 〈0.001）.There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group （P〉0.05）.Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group （P〉0.05）.Conclusions A nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor.HSP70 may be target of leptin in breast cancer.Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.展开更多
The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-P...The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that： （1） Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem- brane of cytotrophoblast; （2） Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly （P〈0.01）; （3） After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls （P〈0.05）. It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro- phoblast invasion and migration activity.展开更多
To observe the effect of growth hormone on serum leptin levels, serum leptin concentrations were measured by enzyme immunoassay in 12 prebutal children with growth hormone deficiency 1, 3 and 6 months before and afte...To observe the effect of growth hormone on serum leptin levels, serum leptin concentrations were measured by enzyme immunoassay in 12 prebutal children with growth hormone deficiency 1, 3 and 6 months before and after the treatment with recombinant human growth hormone (r hGH). For comparison, 34 normal prepubertal children were also investigated. Relationship between leptin levels and body mass index (BMI) was observed at the same time. Our results showed that serum leptin level in normal prepubertal children was 1.22±0.34 ng/ml; the pretreatment serun leptin levels in GHD children was 3.08±2.41 ng/ml, which was significantly different from those 1, 3 and 6 months after GH treatment (i.e. 1.64±1.37 ng/ml,1.57±1.40 ng/ml and 1.35±0.89 ng/ml respectively) (all P <0.001). Our results suggested that r hGH has a suppressive effect on leptin expression.展开更多
Human leptin expressed by \%E.coli\% had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary ...Human leptin expressed by \%E.coli\% had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas. In this study, human leptin gene about 1.0kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3kb were used to construct a expression vector. Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172bp) and parts of the third exon(including part of the encoding sequences and part of the 3′ untranslated region). The final expression vector was digested with \%Not\%I and a fragment of 7.5kb was collected and dissolved in TE(10 mmol/L Tris\5Cl,pH7.4;0.1mmol/L EDTA) for later microinjection. The concentration of DNA was about 2μg/mL, the copy number in 1mL was about 2.4×10 11 , every 1 to 2pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage. After standard microinjection procedures, 48 live mice were obtained. The tails of the mice were cut(about 0.1g) at four weeks of age, genomic DNA was extracted and digested completely with \%Eco\%RI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48). The two female transgenic mice(2\+# and C\-3) were mated with nontransgenic male mice. The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage. SDS\|PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of nontransgenic mouse at fifth day after parturition as control. SDS\|PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin. The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1～2mg/mL.展开更多
Background It is essential to clarify the interactions of hormones during the progression of human breast cancer. This study examined the effects of exogenous human leptin on estrogen receptor （ER） α and β in huma...Background It is essential to clarify the interactions of hormones during the progression of human breast cancer. This study examined the effects of exogenous human leptin on estrogen receptor （ER） α and β in human breast tumor tissue in a nude mouse xenograft model. Methods We created nude mice xenografts of MCF-7 human breast cancer cells, and randomly divided them into an experimental group and a control group. The mice in experimental group were injected subcutaneously around tumors with human leptin, while the control group were injected with the same dose of normal saline. A real-time RT-PCR assay was developed to quantify the mRNA of ERα, β in the tumor tissues. Western blotting analyses were used to assess the relative quantities of the ERα , β proteins. Results Leptin-treated xenografted nude mice were successfully established. The amount of ERa mRNA was significantly higher in the leptin group than in the control group （P 〈0.01）, while the amount of ERβ mRNA was significantly lower in the leptin group than in the control group （P 〈0.01）. Western blotting analyses revealed that the ERa protein level was significantly higher in the leptin group than in the control group （P 〈0.01）, while the ERβ protein level was significantly lower in the leptin group than in the control group （P 〈0.01）. Conclusions Nude mouse xenograft model can be safely and serviceably treated with human leptin by subcutaneous injections around tumor. ERα, β were both targets of leptin in breast cancer. Leptin can up-regulate the expression of ERa and down-regulate the expression of the ERβ in human breast tumor.展开更多
基金This study was supported by a grant from the National Natural Science Foundation of China （No.30772121）.
文摘Background It is important to identify the multiple sites of leptin activity in obese women with breast cancer.In this study,we examined the effect of exogenous human leptin on heat shock protein 70 （HSP70） expression in MCF-7 human breast cancer cells and in a breast carcinoma xenograft model of nude mice.Methods We cultured MCF-7 human breast cancer cells and established nude mice bearing xenograffs of these cells,and randomly divided them into experimental and control groups.The experimental group was treated with human leptin,while the control group was treated with the same volume of normal saline.A real-time reverse transcriptase-polymerase chain reaction （RT-PCR） assay was developed to quantify the mRNA expression of HSP70 in the MCF-7 human breast cancer cells and in tumor tissues.Western blotting analysis was applied to quantify the protein expression of HSP70 in the MCF-7 cells.Immunohistochemical staining was done to assess the positive rate of HSP70 expression in the tumor tissues.Results Leptin activated HSP70 in a dose-dependent manner in vitro：leptin upregulated significantly the expression of HSP70 at mRNA and protein levels in MCF-7 human breast cancer cells （P 〈0.001）.There was no significant difference in expression of HSP70 mRNA in the implanted tumors between the leptin-treated group and the control group （P〉0.05）.Immunohistochemical staining revealed no significant difference in tumor HSP70 expression between the leptin-treated group and the control group （P〉0.05）.Conclusions A nude mouse xenograft model can be safely and efficiently treated with human leptin by subcutaneous injections around the tumor.HSP70 may be target of leptin in breast cancer.Leptin can significantly upregulate the expression of HSP70 in a dose-dependent manner in vitro.
文摘The effects of leptin on cytotrophoblast proliferation and invasion activity were investigated. Immunohistochemistry was used to determine the placental expression of leptin in first-trimester pregnancy. By using RT-PCR and quantitative real-time PCR, the expression of leptin in cytotrophoblast and the effect of leptin on cytotrophoblast secretion were detected. The potential of cell proliferation, invasiveness and migration was assessed by MTT, Transwell invasion assay and migration assay respectively when the cytotrophoblast was cultured with different concentrations of leptin. The results showed that： （1） Leptin was distributed diffusely around cell membrane, in cytoplasma, and on nuclear mem- brane of cytotrophoblast; （2） Leptin mRNA was expressed in cytotrophoblast. Ten ng/mL leptin could promote the secretion of cytotrophoblast significantly （P〈0.01）; （3） After culture with different concentrations of leptin for 24 h or longer, the proliferation of cytotrophoblast was inhibited, while in 24 h leptin could promote cytotrophoblast invasion and migration. Leptin at a concentration of 500 ng/mL could promote cytotrophoblast invasiveness and migration significantly as compared with controls （P〈0.05）. It was suggested that leptin could inhibit cytotrophoblast proliferation, and promote cytotro- phoblast invasion and migration activity.
文摘To observe the effect of growth hormone on serum leptin levels, serum leptin concentrations were measured by enzyme immunoassay in 12 prebutal children with growth hormone deficiency 1, 3 and 6 months before and after the treatment with recombinant human growth hormone (r hGH). For comparison, 34 normal prepubertal children were also investigated. Relationship between leptin levels and body mass index (BMI) was observed at the same time. Our results showed that serum leptin level in normal prepubertal children was 1.22±0.34 ng/ml; the pretreatment serun leptin levels in GHD children was 3.08±2.41 ng/ml, which was significantly different from those 1, 3 and 6 months after GH treatment (i.e. 1.64±1.37 ng/ml,1.57±1.40 ng/ml and 1.35±0.89 ng/ml respectively) (all P <0.001). Our results suggested that r hGH has a suppressive effect on leptin expression.
文摘Human leptin expressed by \%E.coli\% had been used to treat human obesity in American and scientists had achieved good effects, the researchers here wanted to know whether human leptin can be expressed in the mammary glands of transgenic animas. In this study, human leptin gene about 1.0kb, the terminator of rabbit whey acid protein gene (rWAP) about 0.2kb and the promoter including the distal upstream region and part of the first exon of rWAP gene about 6.3kb were used to construct a expression vector. Before we did the subclonings, the sequences of the human leptin gene were sequenced by ABI377 DNA Sequencer, the results showed that the fragment of human leptin gene included the last nine base pairs of the first exon, the complete sequences of the second exon(172bp) and parts of the third exon(including part of the encoding sequences and part of the 3′ untranslated region). The final expression vector was digested with \%Not\%I and a fragment of 7.5kb was collected and dissolved in TE(10 mmol/L Tris\5Cl,pH7.4;0.1mmol/L EDTA) for later microinjection. The concentration of DNA was about 2μg/mL, the copy number in 1mL was about 2.4×10 11 , every 1 to 2pL of the prepared DNA solution was microinjected into the mouse embryos at pronucleus stage. After standard microinjection procedures, 48 live mice were obtained. The tails of the mice were cut(about 0.1g) at four weeks of age, genomic DNA was extracted and digested completely with \%Eco\%RI, two were confirmed to be transgenic mice(both were female) by Southern hybridization using DIG labeled human leptin gene as probe, transgenic rate among the mice born was about 4% (2/48). The two female transgenic mice(2\+# and C\-3) were mated with nontransgenic male mice. The two founder transgenic mice were segregated with their baby mice for at least three hours at the fifth day after parturition and were milked by intraperitoneal injection of 0.3 IU of oxytocin and udder massage. SDS\|PAGE was used to analyze whether there were expression of human leptin in the milk of the two founder transgenic mice with the milk of nontransgenic mouse at fifth day after parturition as control. SDS\|PAGE results showed that compared with the control there was a new band in both of the founder transgenic mice milk, and its molecular weight was about 16 kD, which was quite similar with that of the human leptin. The researchers estimated that the expression level of this protein in the milk of the transgenic mice was about 1～2mg/mL.
基金This study was supported by a grant from the National Natural Science Foundation of China （No. 30772121）.
文摘Background It is essential to clarify the interactions of hormones during the progression of human breast cancer. This study examined the effects of exogenous human leptin on estrogen receptor （ER） α and β in human breast tumor tissue in a nude mouse xenograft model. Methods We created nude mice xenografts of MCF-7 human breast cancer cells, and randomly divided them into an experimental group and a control group. The mice in experimental group were injected subcutaneously around tumors with human leptin, while the control group were injected with the same dose of normal saline. A real-time RT-PCR assay was developed to quantify the mRNA of ERα, β in the tumor tissues. Western blotting analyses were used to assess the relative quantities of the ERα , β proteins. Results Leptin-treated xenografted nude mice were successfully established. The amount of ERa mRNA was significantly higher in the leptin group than in the control group （P 〈0.01）, while the amount of ERβ mRNA was significantly lower in the leptin group than in the control group （P 〈0.01）. Western blotting analyses revealed that the ERa protein level was significantly higher in the leptin group than in the control group （P 〈0.01）, while the ERβ protein level was significantly lower in the leptin group than in the control group （P 〈0.01）. Conclusions Nude mouse xenograft model can be safely and serviceably treated with human leptin by subcutaneous injections around tumor. ERα, β were both targets of leptin in breast cancer. Leptin can up-regulate the expression of ERa and down-regulate the expression of the ERβ in human breast tumor.
