CN104357388A - Red cell membrane rupturing method - Google Patents
Red cell membrane rupturing method Download PDFInfo
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- CN104357388A CN104357388A CN201410696929.2A CN201410696929A CN104357388A CN 104357388 A CN104357388 A CN 104357388A CN 201410696929 A CN201410696929 A CN 201410696929A CN 104357388 A CN104357388 A CN 104357388A
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Abstract
The invention relates to a red cell membrane rupturing method. The red cell membrane rupturing method is characterized by comprising the following steps: taking an appropriate amount of human blood and adding normal saline (0.9% NaCl solution) the volume of which is 3-6 times that of the human body, centrifuging under the conditions of 4 DEG C and 2800rpm for 15-30 minutes to obtain supernate containing leukocytes, plasma proteins and blood platelets, squeezing out the supernate, remaining the packed red cells of the lower layer, repeating the washing procedure 3-5 times until the supernate is basically colorless, removing other substances except for the red cells to the utmost extent to obtain clean packed red cells, taking a certain amount of packed red cells and adding isometric sterilizing high-purity deionized water for pre-swelling for 5-15 minutes, and finally, adding water the volume of which twice that of red cells subjected to pre-swelling to perform low permeation for 10-20 minutes, centrifuging for 30-60 minutes at the rotating speed of 10000, thereby obtaining the supernate which is a hemoglobin solution.
Description
(1) technical field:
The present invention relates to biomedicine field, particularly a kind of red corpuscle rupture of membranes method.
(2) background technology:
In medical aid, very important effect has been played to input the tradition blood transfusion mode that whole blood is Main Means, but simultaneously himself also there are the various shortcomings being difficult to overcome, be mainly manifested in: need to carry out crossmatch before input, there is the danger of multiple viral cross infection, blood source is nervous, storage period is shorter and need special facility.In order to solve above-mentioned contradiction, people just start the research about red blood cell substitute as far back as 19 end of the centurys.Red blood cell substitute refers to have and expands blood volume, maintain extra vascular osmotic balance and have oxygen carrier function, in order to replace the general name of the various oxygen carrier of HRBC.The development of blood substitute experienced by from the blood vessel expander at first without oxygen carrier function, as: hypertonic salt solution, hydroxyethyl starch solution, gelatin etc. have the various carrier solns of function of carrying oxygen up till now.The various product based on oxyphorase developed at present compares with whole blood and has plurality of advantages, as: wide material sources, safety clean, can preserving for a long time, inputting before human body without the need to carrying out crossmatch, the first aid of the patient that is specially adapted to bleed profusely in war, natural disaster and other accidents.Oxyphorase is the material in human body, and its solution has more superiority as red blood cell substitute than chemosynthesis material.
Have 5 products based on oxyphorase at present complete or close to completing III phase clinical study, achieve obvious progress.But there is such as vasoconstriction, elevation of blood pressure all in varying degrees in these product application, the toxic side effect such as impaired organ such as heart, kidney, liver when clinical.In general, the source of the product based on oxyphorase is the expired blood of blood bank, discharges oxyphorase by after red corpuscle rupture of membranes, carries out purifying afterwards, modification obtains goods to oxyphorase.After rupture of membranes, require that in purified hemoglobin, phosphatide residual quantity is very low, otherwise untoward reaction can be caused in follow-up animal, clinical trial, therefore require oxyphorase release as far as possible when rupture of membranes, but film again can not be broken, namely erythrocyte membrane forms complete ghost cell, instead of is fragmented into fragment and is suspended in hemoglobin solutions.
(3) summary of the invention:
The object of the present invention is to provide a kind of red corpuscle rupture of membranes method, it is for above-mentioned situation, a kind of easy and simple to handle, with low cost, efficient rupture of membranes technique of invention.
Technical scheme of the present invention: a kind of red corpuscle rupture of membranes method, is characterized in that it comprises the following steps:
1, the physiological saline (0.9%NaCl solution) that appropriate human blood adds 3-6 times of volume is got, at 4 DEG C, centrifugal under the condition of 2800 turns, 15-30 minute.Obtain containing white corpuscle, plasma proteins and hematoblastic supernatant liquor are also extruded, and retain a layer packed red cells, repeat above-mentioned washing process 3-5 time with, until supernatant liquid is substantially colourless, to remove other materials beyond red corpuscle to greatest extent, obtain clean packed red cells.
2, get a certain amount of packed red cells, add isopyknic sterilizing high purity deionized water and carry out 5-15 minute swollen in advance, finally add the hypotonic 10-20 minute of water of 2 times of volumes, centrifugal 30-60 minute under 10000 rotating speeds, centrifuged supernatant is hemoglobin solutions.
(4) embodiment:
Below the form by embodiment is described in further detail foregoing of the present invention again, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only confined to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1 washes blood
Get the physiological saline (0.9%NaCl solution) that appropriate human blood adds 3-6 times of volume, at 4 DEG C, centrifugal under the condition of 2800 turns, 15-30 minute.Obtain containing white corpuscle, plasma proteins and hematoblastic supernatant liquor are also extruded, and retain a layer packed red cells, repeat above-mentioned washing process 3-5 time with, until supernatant liquid is substantially colourless, to remove other materials beyond red corpuscle to greatest extent, obtain clean packed red cells.
Embodiment 2 is swollen in advance
Get a certain amount of packed red cells, add isopyknic sterilizing high purity deionized water and carry out swollen in advance, the pre-swollen time is decided to be 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30 minutes, respectively at observing different pre-swollen time cellular form under 400 power microscopes, determine the best pre-swollen time according to the mellow and full degree of cell.
The hypotonic rupture of membranes of embodiment 3
In pre-swollen good red corpuscle, add the hypotonic rupture of membranes of water of 2 times of volumes, the time is decided to be 3, and 6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,50 minutes, respectively at observing different rupture of membranes time cellular form under 400 power microscopes, determine the best rupture of membranes time according to the complexity that the concentration of the level of breakage of cell, purified hemoglobin, rupture of membranes liquid filter.Centrifugal 30-60 minute under 10000 rotating speeds more afterwards, centrifuged supernatant is hemoglobin solutions.
Claims (3)
1. a red corpuscle rupture of membranes method, is characterized in that it comprises the following steps: get the physiological saline (0.9%NaCl solution) that appropriate human blood adds 3-6 times of volume, at 4 DEG C, centrifugal under the condition of 2800 turns, 15-30 minute; Obtain containing white corpuscle, plasma proteins and hematoblastic supernatant liquor are also extruded, and retain a layer packed red cells, repeat above-mentioned washing process 3-5 time with, until supernatant liquid is substantially colourless, to remove other materials beyond red corpuscle to greatest extent, obtain clean packed red cells; Get a certain amount of packed red cells, add isopyknic sterilizing high purity deionized water and carry out swollen in advance, finally add the hypotonic 10-20 minute of water of 2 times of volumes, centrifugal 30-60 minute under 10000 rotating speeds, centrifuged supernatant is hemoglobin solutions.
2. a kind of red corpuscle rupture of membranes method according to claim 1, the time swollen is in advance 5-15 minute.
3. a kind of red corpuscle rupture of membranes method according to claim 1, the time of hypotonic rupture of membranes is 10-20 minute.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410696929.2A CN104357388A (en) | 2014-11-25 | 2014-11-25 | Red cell membrane rupturing method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410696929.2A CN104357388A (en) | 2014-11-25 | 2014-11-25 | Red cell membrane rupturing method |
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| CN104357388A true CN104357388A (en) | 2015-02-18 |
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| CN201410696929.2A Pending CN104357388A (en) | 2014-11-25 | 2014-11-25 | Red cell membrane rupturing method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966266A (en) * | 2019-02-26 | 2019-07-05 | 南华大学 | The polymer matrix medicament-carried nano compound system and preparation method thereof of erythrocyte membrane package |
| CN110398401A (en) * | 2019-07-12 | 2019-11-01 | 上海晶铸生物科技有限公司 | A kind of method of Thinprep pap test film-making |
-
2014
- 2014-11-25 CN CN201410696929.2A patent/CN104357388A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966266A (en) * | 2019-02-26 | 2019-07-05 | 南华大学 | The polymer matrix medicament-carried nano compound system and preparation method thereof of erythrocyte membrane package |
| CN110398401A (en) * | 2019-07-12 | 2019-11-01 | 上海晶铸生物科技有限公司 | A kind of method of Thinprep pap test film-making |
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Application publication date: 20150218 |
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