Polypeptides regulating sugar metabolism and uses thereof
A technology of glucose metabolism and peptides, applied in the field of biomedicine, can solve problems such as deficiency and insulin resistance
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[0082] DMEM medium was purchased from Sigma, wherein every 500ml medium supplemented with 10% FBS (fetal bovine serum), 1% non-essential amino acids, 1g glucose, 0.75g sodium bicarbonate, 0.1g bovine serum albumin and 1.5ml HEPES (4 -Hydroxyethylpiperazineethanesulfonic acid).
[0083] ISAPs 1 The sequence is Tyr-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Thr (SEQ ID NO. 22). ISAPs 2 The sequence is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO.2). ISAPs 3 The sequence of is Tyr-Leu-Tyr-Gln-Trp-Leu-Gly-Ala-Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro-Arg (SEQ ID NO.3). ISAPs 4 The sequence is Ser-Val-Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO.15). ISAPs 5 The sequence is Pro-Ser-Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO.16). ISAPs 6 The sequence is Pro-Asp-Pro-Leu-Glu-Pro (SEQ ID NO.14).
[0084] βTC-6 was purchased from the American Type Culture Collection (ATCC). This cell line is derived from mouse pancreatic cancer and has introd...
Embodiment 1
[0087] ISAPs 1 function of
[0088] 1. ISAPs 1 Effects on βTC-6 cells.
[0089] The experimental steps are as follows:
[0090] 1. Cell plating: Dilute the βTC-6 cell suspension at 1.6×10 5 / mL density plated in 24-well plates, 0.5 mL per well, at 37 °C, 5% CO 2 Incubate for 24 hours.
[0091] 2. Dilute ISAP with sugar-free DMEM 1 Dilute to 2nmol / L, 200pmol / L, 20pmol / L, 2pmol / L respectively and store in ice bath.
[0092] 3. Perform starvation pretreatment on βTC-6 cells, using KRBB buffer (129g NaCl, 5g NaHCO 3 ,4.8gKCl,1.2gKH 2 PO 4 ,1.2g MgSO 4 ,2.5g CaCl 2 , 10mM HEPES and 0.1% BSA at pH 7.4) for 4 hours.
[0093]4. The cell culture medium was discarded, and ISAP containing different concentrations were added to the experimental group and the positive control group respectively. 1 and glucose culture solution, the negative control group only added medium (blank), at 37 ° C, 5% CO 2 Neutralize for 1 hour.
[0094] 5. Wash the cells at the bottom of the plate ...
Embodiment 2
[0123] ISAPs 2 The function ISAP 2 Binding to human GPRC6A
[0124] 2.1 Overexpression of hGPRC6A in Hela cells 1) Cell plating: Hela cell suspension was mixed with 1.6×10 5 / mL density plated in 6-well plates, 2 mL per well of DMEM medium, at 37 °C, 5% CO 2Incubate for 24 hours.
[0125] 2) Using Lipofectamine2000 (Invitrogen), the hGPRC6 overexpression vector (pReceiver-M61) was transfected into Hela cells according to the manufacturer's instructions, and the normal medium was replaced 4 hours after transfection.
[0126] 3) 48 hours after transfection, discard the medium, wash the cells at the bottom of the plate twice with sterile PBS, add 300 μL of Trizol solution, extract cellular RNA according to the instructions of RNAiso Plus (TaKaRa), after DNase treatment, use SuperScript T (Invitrogen, Canada) kit was reverse transcribed into cDNA by DNAEngine. Using cDNA as a template, use SYBRGr ee The n method was used to monitor and analyze the fluorescent PCR products at...
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