As early as 1930, Smith discovered that the thymus began to shrink after the pituitary was removed from the rat [1]. Later, there is growing evidence that growth hormone (GH) is closely related to immune cells: (1) GH promotes T lymphocyte development, especially during thymus development; (2) in bovine GH In transgenic mice, overexpression of GH results in a significant increase in the number of hematopoietic progenitors; (3) the addition of specific antibodies or antisense oligonucleotides to GH can prevent the proliferation of lymphocytes by GH. The laboratory has demonstrated that the nucleotide sequence of GH cD2 NA and its promoter in human lymphocytes is identical to that of the pituitary [2,3]. To investigate the regulation of GH on T lymphocyte function, this study constructed a GH cDNA expression plasmid, which was then transfected into the T lymphocyte line, J urkat E621, to observe the overexpression of endogenous GH and to add it. The effect of recombinant human GH on the expression of interleukin 2 receptor (IL22R) and interleukin 2 (IL22) and interferon-gamma (IFN2γ) expressed by T lymphocytes.
1 Materials and methods
111 cells, strains and plasmids T lymphocytes were Jurkat E621 (purchased from ATCC, USA). E1 coli DH5α is a strain for preservation in this room. The PUC192GH cDNA contains a 804 bp GH cDNA and is constructed for preservation in this room. PcDNA311 ( + ) is a gene expression vector for eukaryotic cells (a gift from Professor Zheng Dexian from the Department of Biochemistry, Chinese Academy of Medical Sciences).
112 Enzymes, Cell Culture and Transfection Reagents for Cloning Plasmids Restriction Endonucleases BamHI, EcoRI, T4DNA Ligase, Wizard PCR Product Purification System Kit was purchased from Promega. RP2 MI1640 medium, OPTI2MEM culture medium for transfection, and AIMV serum-free medium are products of GIBCO. Fetal bovine serum (FCS) is a product of the Institute of Hematology, Chinese Academy of Medical Sciences. L2 glutamine, a product of Sigma. Liposomal Transfection Reagent DMRIE2C Regent is a product of GIBCO. Anti-human GH serum is made by our own.
113 Kit for detection of IL22R, IL22, IFN2γ and GH Flow cytometry detection IL22R The EL ISA kit for detection of monoclonal antibodies, IL22 and IFN2γ with anti-CD25 fluorescent label and negative control is a product of IMMUNOTEC, France. GH Detection The EL ISA kit is self-made in this room.
Construction of 114 GH expression plasmid PcDNA2GH cDNA As shown in Fig. 1, a GH expression plasmid PcDNA2GH cDNA containing a cytomegalovirus promoter (CMV) was constructed.
115 DMRIE2C Regent method for transfecting Jurkat cells Jurkat cells with good growth were centrifuged at 800 r/min for 5 min, washed once with serum-free DMEM medium, then resuspended in OPTI2MEM medium, and adjusted for cell concentration per ml. 1 × 107. Prepare A and B solution: Take 500μl OPTI2MEM medium and 4μl DM2 RIE2C Regent into 115ml sterile Eppendorf tube and mix gently into solution A; take another 115ml sterile Eppendorf tube, add 500μl OPTI2MEM medium and transfer Dye the plasmid (115 μg of the plasmid to be transfected) and mix it to form B solution. Mix the A and B liquids into a six-well culture dish and place them at room temperature for 45 min before adding 200 μl of Jurkat cells (1
×106), placed in a CO2 incubator, cultured at 37 °C, 5 % CO2 for 5 h, then add 2 ml of serum-free AIMM medium per well.
116 Overexpression of GH on the expression of IL22R and IL2 2 and IFN2γ secretion in Jurkat cells In this experiment, three groups were transfected with PcDNA2GH cDNA and only PHA was added to a final concentration of 30 μg/ml, and the other group was transfected with plasmid. PHA and anti-GH antibody (1:1 000) were added, and Jurkat cells with PHA only in the non-transfected plasmid were used as control group, cultured at 37 °C, 5% CO2 for 72 h, and then the cell culture supernatant was collected to detect GH, IL22 and At the concentration of IFN2γ, cells were immediately assayed for the number of IL22R-expressing cells by flow cytometry.
117 Effect of exogenous GH on IL22R expression and secretion of IL22 and IFN2γ in Jurkat cells Jurkat cells with good growth state were collected, and the concentration of cells was adjusted to 5 × 105 cells per ml by adding serum-free medium AIMV to 1 ml per well. The cells were added to a 24-well culture plate and divided into 5 groups. Each group was added with PHA to a final concentration of 30 mg/L. Group 1 was the control group and the other 4 groups were the experimental groups. Humatrope GH was added to the final concentration of 1, 3, and 25 respectively. After 100 h/L, 48 h, the cells were collected for flow cytometry to detect IL22R, and the culture supernatant was stored in a -70 °C refrigerator for detection of IL22 and IFN2γ.
118 Flow cytometry to detect samples of IL22R Prepare Jurkat cells to be collected, wash cells once with 0101 mol/L p H714 in PBS, adjust the cell concentration to 106 cells/ml, and take 100 μl of each sample into the measuring tube. Then, 10 μl of monoclonal antibody against CD252FITC was added, and the same sample was added to the mouse IgG12FITC as a negative control. After standing for 15 min at room temperature in the dark, the cells were washed twice with PBS, and then up-flow cytometry was performed, and 10 000 cells were counted at the time of detection.
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119 Detection of GH, IL22 and IFN2γ in cell culture supernatants The experiment was performed using the EL ISA method according to the instructions.
1110 Calculation of results and statistical analysis Each experiment was repeated 3 times. In the statistical analysis of the results, the average measured value of the control group was set to 1. Each experimental group was compared with it to obtain a relative value, and the test was performed by t test. For comparison, it is meaningful to have P < 0105.
2 results
Identification of 211 PcDNA2GH cDNA expression plasmid The constructed plasmid was digested with BamHI/EcoRI, and a band of about 800 bp was excised by 1% agarose electrophoresis. At the same time, 115 μg of the purified plasmid was transfected into Jurkat cell culture solution. The concentration of GH was measured, and the GH content in the Jurkat cell culture medium of the transfected plasmid group was about 014 μg/L, and the GH content in the Jurkat cell culture solution in the non-transfected group was not detected. That is, the expression plasmid was successfully constructed.
212 overexpressed endogenous GH on lymphocyte surface IL22R
Expression and effects of IL22 and IFN2γ secretion (Fig. 2)
The effect of overexpressed endogenous GH on IL22R level The expression of IL22R on the surface of Jurkat cells transfected with GH cDNA was significantly higher than that of the control group, which was 1140 times that of the control group (P
< 0105), and the addition of anti-GH antibody, the expression of IL2 2R on the surface of Jurkat cells was significantly lower than that of the GH cDNA group (P < 0105) (Table 1).
21212 Overexpression of endogenous GH secreted IL22 in Jurkat cells. The secretion of IL22 from Jurkat cells transfected with GH cDNA was significantly increased compared with the control group, which was 1160-fold higher than that of the control group (P < 0105), while transfected with GH cDNA. The secretion of IL22 was significantly reduced in the Jurkat cells after addition of anti-GH antibody compared with the GH cDNA group (P < 0105) (Table 2).
The effect of overexpressed endogenous GH on the secretion of IFN2γ by Jurkat cells showed that the secretion of IFN2γ from Jurkat cells transfected with GH cDNA was increased by 1198-fold compared with the control group (P < 0101), while transfected with GH cDNA. After addition of anti-GH antibody to Jurkat cell culture medium, IFN2γ secretion was significantly reduced compared with Jurkat cells in the GH cDNA group (P < 0101) (Table 3).
213 Effects of exogenous GH on lymphocyte function (Fig. 3)
21311 Effect of exogenous GH on IL22R levels The expression of IL22R on the surface of Jurkat cells was detected by flow cytometry. It was found that exogenous GH 1μg/L promoted the expression of IL22R, which was 1140 times of the control group (P < 0101). When the GH concentration was greater than 3 μg/L, the promotion of IL22R expression by GH increased with the dose of GH. And weakened (Table 4).
21312 Effect of exogenous GH on IL22 level The concentration of IL22 in Jurkat cell culture medium was detected by EL ISA method. The results showed that exogenous GH could be promoted at 3～10 0μg/L.
21313 Effect of exogenous GH on secretion of IFN2γ from Jurkat cells The results showed that 3 and 25 μg/L GH significantly increased the secretion of IFN2γ from Jurkat cells (P < 0105), which was attenuated with the dose of GH (Table 6).
3 Discussion
HGH is a polypeptide containing 191 amino acids. It acts as a major regulator of cell growth and stimulates the development and enlargement of all tissues and increases the size and number of somatic cells. However, many recent and in vitro studies have shown that GH can affect the function of T cells and promote the development of T cells in the thymus [4]; induce the production of IL21 and IL2 2; enhance cytotoxic T cells (Tc), natural killer cells ( NK), delayed-type hypersensitivity T cell (TDTH) activity and promotion of antibody production against thymus-dependent (TD) antigens. It is also believed that under physiological conditions, GH plays an important regulatory role in the function of immune cells. How GH regulates lymphocyte function is not well understood. In particular, how saizen GH overexpressing GH affects its function has not been reported. Therefore, we studied the effects of exogenous GH on T lymphocyte function and found that GH is T. The function of lymphocytes is significantly promoted, which is manifested by increased expression of IL22R and increased secretion of IL2 2 and IFN2γ. The effect of GH on the function of T lymphocytes was observed to be dependent on the dose when observing the action of exogenous GH. Different doses of GH promoted the expression of IL22R in PHA-activated Jurkat cells at a dose of 1 μg/L. Maximize, as the dose of GH increases, the promotion of IL22R expression in activated Jurkat cells is diminished. The effect of GH on the secretion of IL22 by activated Jurkat cells is exactly the opposite. As the dose of GH increases, it promotes the activation of Jurkat cells to secrete IL22. This may be due to the high secretion of GH to increase the secretion of IL22 secreted by activated Jurkat cells, which leads to the down-regulation of IL22R expression in Jurkat cells, which reduces the expression of IL22R in Jurkat cells. Studies of GH on the secretion of IFN2γ by activated Jurkat cells showed that the optimal dose was 3 μg/L. When the GH cDNA expression plasmid was transfected into lymphocytes, it was found that overexpression of endogenous GH had no effect on the expression of IL22R on the surface of quiescent Jurkat cells (data not shown), whereas the expression of surface IL22R after PHA activated Jurkat cells Significantly increased, after the addition of anti-GH antibody (1:1 000), the excitatory effect of endogenous GH on IL22R disappeared; endogenous GH also increased the levels of IL22 and IFN2γ secreted by PHA-activated Jurkat cells to 1160 ( 160%) and 1198 (198%), anti-GH antibodies were added to counteract this effect, IL22 and IFN2γ recovered to near control levels (114% and 95%, respectively). These results indicate that GH is closely related to T lymphocytes.
IL22R is a marker of lymphocyte activation. The surface of normal quiescent lymphocytes expresses only a small amount of IL22R. When lymphocytes are activated by antigen or mitogen stimulation, they express abundant IL22R on the surface, and IL22R binds to IL22. After that, lymphocytes can further proliferate directly into effector cells, and they can also secrete various cytokines such as IL22, IFN2γ, TNF, IL24, IL25 and IL26 to regulate cellular immunity and humoral immunity. Thus, IL22R can be seen. The expression is essential for lymphocyte activation. Our studies have shown that both exogenous and endogenous GH can increase the expression of PHA-activated lymphocytes IL22R, IL22 and IFN2γ, and anti-GH antibodies can counteract this effect, indicating that GH regulates lymphocyte function. Autocrine and paracrine GH of lymphocytes play an important role in maintaining the normal function of lymphocytes. These results make us more aware of the close relationship between the neuroendocrine system and the immune system, and also provide a theoretical basis for the more widely used clinical application of recombinant human GH.