CN102716505A - Method for disinfecting in-vitro skin by virtue of ultraviolet B and product thereof - Google Patents

Abstract

The invention discloses a method for disinfecting an in-vitro skin by virtue of an ultraviolet B, specifically comprising the following step of: irradiating the in-vitro skin by an ultraviolet B having a voltage of 180 to 240 V and an irradiation dose of 100 to 400 J/cm. The method is simple and controllable in condition; the disinfected skin is high in the activity, low in carrier volume, and strong in antigenicity; and the prepared disinfected skin can be used for replacing a dressing for treating a large-area burn of an autologous skin.

Description

Utilize ultraviolet B radiation to isolated skin disinfectant method and products thereof

Technical field

The invention belongs to a kind of sterilization method, be specifically related to utilize ultraviolet B radiation, also relate to the product that makes with this method the isolated skin disinfectant method.

Background technology

Allosome and xenogenesis skin are the main dressing that substitutes autologous skin treatment large-area burns clinically; But xenogenesis and heterogenous skin antigenicity are strong; Particularly fresh skin also has a large amount of fungi pollutions, and the strong immunization rejection after the skin transplantation tends to the necrosis that causes skin too early.(UltravioletRadiationB UVB), is to arrive the main spectrum that causes the body biological effect in the ultraviolet of earth surface, wavelength 280-320nm to ultraviolet B radiation.The narrow spectrum ultraviolet B radiation (NB-UVB) that shows 311nm after deliberation is in the good unicity of treatment psoriasis tool; Erythematous response, pigmentation are slight; And curative effect is reliable, can produce optimum therapeuticing effect, and simple NB-UVB irradiation treatment psoriasis effective percentage can reach 73.3%; And the UVB of 290-300nm wavelength can cause skin burn (erythemal effect and keratinocyte are downright bad), and does not have therapeutical effect.At present, it is blank to utilize ultraviolet B radiation that the isolated skin sterilization is also handled, and how to reduce the immunogenic while of isolated skin, and content of molds is few, can keep the isolated skin activity higher again.It is problem demanding prompt solution in the skin transplantation.

Summary of the invention

In view of this; One of the object of the invention is to provide and utilizes ultraviolet B radiation to the isolated skin disinfectant method, and is simple to operation, and condition is easy to control; Less to the isolated skin activity influence, and can effectively control the content of molds of isolated skin and reduce immunogenicity.

For realizing the foregoing invention purpose, technical scheme is:

Utilize ultraviolet B radiation to the isolated skin disinfectant method, it is 180 ~ 240V that isolated skin is used voltage, and exposure dose is 100 ~ 400J/cm ²Ultraviolet B radiation irradiation.

Preferably, it is 0 ~ 25 ℃ that said isolated skin places temperature, and humidity is under the environment of 40-70%.

Preferred, it is 25 ℃ that said isolated skin places temperature, and humidity is under 60% environment, uses voltage to be 220V, and exposure dose is the ultraviolet B radiation irradiation of 300J/cm.

Preferred, also comprise the isolated skin pretreatment before the ripple ultraviolet radiation, be specially isolated skin is processed the material that thickness is 1.0mm.

Isolated skin can be selected acceptable clinically skin for use among the present invention, and preferred isolated skin has wide material sources, the characteristics that cost is low for the Corii Sus domestica that exsomatizes.

Two of the object of the invention is to provide by the isolated skin of ultraviolet B radiation to isolated skin sterilization preparation, and its immunogenicity is low, and content of molds is few, and skin activity is high.

For realizing the foregoing invention purpose, technical scheme is:

The said isolated skin that utilizes ultraviolet B radiation to the preparation of isolated skin disinfectant method.

Beneficial effect of the present invention is: the ultraviolet B radiation that utilizes disclosed by the invention is to the isolated skin disinfectant method; Simple to operate, condition is easy to control, and is less to the isolated skin activity influence; And the isolated skin content of molds that makes is few; Immunogenicity is low, can be used for substituting clinically the dressing of autologous skin treatment large-area burns, is with a wide range of applications.

The specific embodiment

Below will carry out detailed description to the preferred embodiments of the present invention with reference to embodiment.The experimental technique of unreceipted actual conditions in the preferred embodiment is usually according to the normal condition or the condition of advising according to manufacturer.

One, sterilization method

Utilize ultraviolet B radiation stereo Corii Sus domestica method of disinfecting, concrete steps are following:

A system skin

Get fresh porcine skin, processing thickness then is the stripped Corii Sus domestica of 0.8-1.0mm, and stripping and slicing is subsequent use;

The B irradiation

The stripped Corii Sus domestica of processing of step A is positioned over ultraviolet B radiation (UVB) calorstat, and the distance that keeps Corii Sus domestica and UVB fluorescent tube is handled the Corii Sus domestica of must sterilizing less than 20cm according to the orthogonal test of table 1 design to Corii Sus domestica.Get fresh porcine skin, handle the negative contrast of skin, shine positive contrast with 25kGYCo^60 without UVB.25kGYCo^60 representes the dosage with 25kGY cobalt-60; In the RADIATION PROCESSING process, receive to shine the energy that the material unit mass is absorbed, iu is defined as gray(Gy) (Gy); 1 gray(Gy) is represented 1 kilogram of (Kg) material absorbing erg-ten (J) energy, 1 Gy=1 J/ Kg; Kilogray (KGy) expression absorbed dose commonly used in the RADIATION PROCESSING.

Table 1. orthogonal experiment method UVB irradiation Corii Sus domestica factor and water-glass

Remarks: V-voltage; T-temperature; S-humidity; Z-exposure dose

Handle the back and measure each index of skin, be respectively UVB epiderm skin cytositimulation lymphopoiesis suppression ratio (YZL); The UVB irradiation is to skin LC quantity decline percentage rate (LC); UVB is to skin content of molds antibacterial decline percentage rate (XJ) and the postradiation skin activity percentage rate of UVB (HX).

Two, lymphocyte proliferation assay

1) lymphocytic separation

Lymphocyte separation medium (grind open up bio tech ltd available from Shanghai) and healthy subjects whole blood are successively added centrifuge tube for 1:1 by volume, are that 25 ℃, rotating speed are under the 3000rpm condition centrifugal 20 minutes in temperature; Draw the intermediate layer buffy coat, and clean 3 times, carry out viable count then, and use mass fraction to adjust cell concentrations to l * l0 as 10%CS-RPMI 1640 with lymphocyte separation medium ⁶Cells/ml.

2) separation of epidermis cell

The Corii Sus domestica of will sterilizing is cut into 0.3cm * 2cm leather strap, and the adding mass fraction is that the volume ratio of 0.25% the trypsin trypsin and the Corii Sus domestica of sterilizing is 5:l), place 4 ℃ of refrigerators digestion 15 hours, take epidermis gently off with tweezers.Shredding epidermis in temperature is under 37 ℃ of conditions; Using mass fraction is that heat digested 0.5 hour in the 0.25% trypsin shaking table; Add the serum that is equivalent to Digestive system 1/10 volume and stop digestion; At rotating speed is under the 1000rpm condition centrifugal 5 minutes, carries out the living cells cell counting then, and to use mass fraction be that 10% CS-RPMI, 1640 adjustment cell concentrations are to l * l0 ⁶Cells/mL.

3) making sheet

Every hole adds l * 10 in 96 orifice plates ⁵Cells lymphocyte and l * 10 ⁴The cells epidermis cell is an experimental group, and blank control group adds l * 10 separately ⁵Cells lymphocyte, matched group add equal number without the UVB isolating l of Corii Sus domestica * 10 of handle exsomatizing ⁴Cells epidermis cell and l * 10 ⁵The cells lymphocyte, all the other are supplied with culture fluid, do 5 multiple holes for every group.Liquid feeding finishes, and moving 96 orifice plates is 37 ℃ in temperature, and volume fraction is 5% CO ₂Hatching 50 hours, is under the 1000rpm condition centrifugal 5 minutes at rotating speed then, and abandoning liquid, to add mass fraction be 0.05% MTT (MTT), 100 μ L; Continue to cultivate 4 hours; After cultivating end, abandon liquid and add 150 μ L dimethyl sulfoxide (DMSO), piping and druming evenly; Treat that purple crystal dissolves fully, putting the flat Tissue Culture Plate in 96 holes is that the 570nm place measures the OD value in the integrated enzyme reaction detector at wavelength.Calculate suppression ratio: suppression ratio=1-(experimental group OD value-blank control group OD value/matched group OD value-blank control group OD value) * 100% according to following formula, the result is as shown in table 2.

Three, ATP enzyme dyeing Lang Gehan cell (LC)

The epidermis that separates the sterilization Corii Sus domestica according to the method described above is an experimental group, and with the stripped Corii Sus domestica separating table skin handled without UVB as matched group.

1) ATP enzyme dyeing

The maleic acid buffer of the epidermis that separates being put into the Tris that contains 30g/L and 29g/L respectively launches (comprising experimental group and matched group), washs 3 times, washs 10 minutes at every turn; Putting into the fixative of the formalin of swollen acid of the diformazan that contains 12.8g/L and 40g/L, is to fix 20 minutes under 4 ℃ of conditions in temperature; The epidermis that fixes is put into the Incubating Solution that contains 0.45g/L ATP disodium and 2.0g/L plumbi nitras, is to hatch 40 minutes under 37 ℃ of conditions in temperature; Hatch the back and uses concentration to wash 3 times, cleaned 10 minutes at every turn as the Tris-maleic acid buffer (pH5.0-8.5) of 0.1mol/L; In containing the ammonium sulfide of 11g/L, developed the color 20 minutes then; Reuse tap water flushing 10 minutes.With corium towards last flattening on microscope slide, after the glycerin gelatine sealing, amplify the size of 100 times of BMDCs (Lang Gehan cell) of observing ATP enzyme stained positive down and distribute, and photomicrography at light microscopic.

2) mensuration of epidermis unit are LC quantity

Under light microscopic amplifies 100 times, select 10 visuals field at random, calculate every mm with the eyepiece grid mircrometer gauge ²ATP enzyme stained positive BMDC (Lang Gehan cell) quantity in the area, UVB shines skin LC quantity decline percentage rate=(matched group LC quantity-experimental group LC quantity)/matched group LC quantity * 100%, and the result is as shown in table 2.

Four, content of molds detects

1) the sterilization Corii Sus domestica is handled

The Corii Sus domestica of will sterilizing adds sterile saline, and the weight of the Corii Sus domestica of wherein sterilizing and the volume ratio of normal saline are 1:2, and the Corii Sus domestica culture dish of will sterilizing then is cut into pasty state with eye scissors with skin graft.

2) bacterium is cultivated

With staphylococcus aureus and the positive contrast of Candida albicans, with the negative contrast of normal saline, concrete grammar is edited with reference to Chinese Pharmacopoeia Commission. three .2005 versions of Pharmacopoeia of People's Republic of China, appendix: 73-84.Fall into inoculation 100 μ L sterilization Corii Sus domestica homogenate on the culture plate, 3 repetitions of each sample at the bactericidal nurishing agar culture medium.Be to cultivate 48 hours under 30 ℃ of conditions in temperature then, carry out colony counting with gel imaging system.With the content of molds of following formula calculating sterilization Corii Sus domestica, cfu/g=bacterium colony number/(the heavy g of skin graft * 100 μ L/ add the amount μ L of normal saline).UVB is to skin content of molds antibacterial decline percentage rate=(positive controls cfu/g-experimental group cfu/g)/positive controls cfu/g * 100%, and the result is as shown in table 2.

The result of table 2, the test of UVB irradiation Corii Sus domestica condition orthogonal experiment method

?	V	T	S	Z	Blank	YZL （%）	LC（%）	XJ （%）	HX （%）
										1	180	0	40	100	1	0.281	0.1472099	0.182168	0.902
2	180	5	50	200	2	0.538	0.30253	0.565851	0.911
										3	180	15	60	300	3	0.710	0.411909	0.702419	0.868
4	180	25	70	400	4	0.665	0.397383	0.662472	0.849
										5	200	0	50	300	4	0.710	0.4118078	0.702246	0.868
6	200	5	40	400	3	0.794	0.4743835	0.790838	0.819
										7	200	15	70	100	2	0.617	0.3232829	0.400052	0.786
8	200	25	60	200	1	0.996	0.7287604	0.998563	0.833
										9	220	0	60	400	2	0.960	0.6103089	1.017438	0.768
10	220	5	70	300	1	0.991	0.5751634	0.980813	0.816
										11	220	15	40	200	4	0.998	0.6096112	0.9993	0.820
12	220	25	50	100	3	0.880	0.4612434	0.570774	0.694
										13	240	0	70	200	3	0.884	0.4969213	0.92944	0.853
14	240	5	60	100	4	0.739	0.3874901	0.479507	0.743
										15	240	15	50	400	1	0.997	0.8512945	0.999845	0.676
16	240	25	40	300	2	0.995	0.8779025	0.99986	0.719

Remarks: V-voltage; T-temperature; S-humidity; Z-exposure dose; YZL-UVB is to epiderm skin cytositimulation lymphopoiesis suppression ratio; LC-UVB shines skin LC quantity decline percentage rate; XJ-UVB is to skin content of molds antibacterial decline percentage rate; The postradiation skin activity percentage rate of HX-UVB.

Five, mtt assay skin activity test experience

Getting fresh untreated Corii Sus domestica is matched group, and the sterilization Corii Sus domestica is an experimental group, uses diameter to be 5 of each sample bark fetching of card punch of 5mm then, and it is 0.05% MTT lmL that every skin adds mass fraction respectively, is that 37 ℃, volume fraction are 5% CO in temperature ₂Cultivated under the condition l hour, and abandoned liquid and add 2.5mL DMSO in room temperature shaking table internal reaction 2 hours, the vibration back adds in 96 orifice plates, 5 multiple holes, the mensuration OD value (OD) in integrated enzyme reaction detector 570nm place.The postradiation skin activity percentage rate of UVB=experimental group OD value/matched group OD value * 100%, the result is as shown in table 2.

Six, carry out range analysis through SPSS software pair of orthogonal experimental data

1. UVB is to the range analysis of the influence of epiderm skin cytositimulation lymphopoiesis suppression ratio, and the result is as shown in table 3.

Table 3, UVB influence result and range analysis to epiderm skin cytositimulation lymphopoiesis suppression ratio

?	V	T	S	Z	Blank
						k1	0.54851	0.708444	0.767089	0.629223	0.816064
k2	0.77908	0.765675	0.781088	0.853942	0.777287
						k3	0.95726	0.830423	0.851058	0.851239	0.816959
k4	0.90363	0.883935	0.789243	0.854074	0.778167
						R	0.40874	0.1754909	0.0839693	0.2248513	0.0396727

Can know by table 3; Voltage (V) is the most obvious to the influence of epiderm skin cytositimulation lymphopoiesis, and exposure dose (Z) is taken second place, and humidity (S) is minimum to the influence of epiderm skin cytositimulation lymphopoiesis; Concrete order is V>Z>T>S is best with V3T4S3Z4.

2. the UVB irradiation is to the range analysis of skin LC quantity decline percentage rate influence, and the result is as shown in table 4.

Table 4, UVB irradiation influence result and range analysis to skin LC quantity decline percentage rate

?	V	T	S	Z	Blank
						k1	0.31476	0.416562	0.5272768	0.3298066	0.5756078
k2	0.48456	0.4348918	0.5067189	0.5344557	0.5285033
						k3	0.56408	0.5490244	0.5346171	0.5691957	0.4611111
k4	0.65340	0.6163223	0.4481876	0.5833425	0.4515719
						R	0.33864	0.1997603	0.0864295	0.2535359	0.1240359

Can be known that by table 4 voltage (V) is the most obvious to skin LC quantity decline influence, exposure dose (Z) is taken second place, and humidity (S) descends to skin LC quantity influences minimum, and specifically order is V>Z>T>S is best with V4T4S3Z3.

3. UVB is to the range analysis of the influence of skin content of molds antibacterial, and is as shown in table 5.

Table 5, UVB are to skin content of molds antibacterial descend percentile result of influence and range analysis

?	V	T	S	Z	Blank
						k1	0.52823	0.7078228	0.7430414	0.4081251	0.7903471
k2	0.72292	0.7042522	0.7096791	0.8732885	0.7414405
						k3	0.89208	0.7754041	0.7994814	0.8463344	0.7483677
k4	0.85216	0.807917	0.7431941	0.8676482	0.7108818
						R	0.36385	0.1036648	0.0898023	0.4651633	0.0794653

Can be known that by table 5 exposure dose (Z) is the most obvious to the influence of skin content of molds, voltage (V) takes second place, and humidity (S) is minimum to the influence of skin content of molds, and concrete order is Z>V>T>S is best with V3T4S3Z2.

4. the range analysis of the postradiation skin activity influence of UVB, the result is as shown in table 6.

Table 6, the postradiation skin activity percentage rate of UVB influence result and range analysis

?	V	T	S	Z	Blank
						k1	0.88246	0.8478613	0.8151432	0.7813042	0.8068101
k2	0.82658	0.8222215	0.7872132	0.8543271	0.7956788
						k3	0.77440	0.7874164	0.8029882	0.8176122	0.808729
k4	0.74776	0.7736946	0.8258492	0.7779504	0.8199759
						R	0.13470	0.0741666	0.038636	0.0763767	0.0242971

Can be known that by table 6 voltage (V) has the greatest impact to skin activity, be exposure dose (Z) secondly, and humidity (S) is minimum to the skin activity influence, and concrete order is V>T>Z>S is best with V1T1S4Z2.

seven, carry out variance analysis through SPSS software pair of orthogonal experimental data.

1. to the variance analysis of UVB to the influence of epiderm skin cytositimulation lymphopoiesis suppression ratio, the result is as shown in table 7.

Table 7, UVB influence result and variance analysis to epiderm skin cytositimulation lymphopoiesis suppression ratio

Can be known that by table 7 UVB has statistical significance to voltage (V), temperature (T) and exposure dose (Z) factor in the influence of epiderm skin cytositimulation lymphopoiesis suppression ratio, the influence of humidity (S) factor does not have statistical significance.

2. the UVB irradiation is as shown in table 8 to the The results of analysis of variance that skin LC quantity decline percentage rate influences the result.

Table 8, UVB irradiation influence result and variance analysis to skin LC quantity decline percentage rate

Can be known by table 8, be evaluation index with LC decline ratio, and model does not have statistical significance.

3. UVB is to the descend percentile result's of influence the variance analysis of skin content of molds antibacterial, and the result is as shown in table 9.

Table 9, UVB are to skin content of molds antibacterial descend percentile result of influence and variance analysis

Can be known by table 9, be that evaluation index voltage (V) and exposure dose (Z) factor have statistical significance with antibacterial decline ratio, and temperature (T) and humidity (S) do not have statistical significance.

4. the postradiation skin activity percentage rate of UVB is influenced result's variance analysis, the result is as shown in table 10.

Table 10, the postradiation skin activity percentage rate of UVB influence result and variance analysis

Can be known by table 10, be evaluation index with the skin activity, and voltage (V), temperature (T), exposure dose (Z) factor have statistical significance, and the influence of humidity (S) factor does not all have statistical significance.

Is basis for estimation with the threshold value of each index and each factor to the significance of the influence of index; Through taking all factors into consideration optimum condition is V3T4S3Z3; Be 220V at voltage promptly, temperature is 25 ℃, and humidity is 60%; The stripped Corii Sus domestica of narrow spectrum ultraviolet B radiation irradiation of 300J/cm can obtain to reduce to greatest extent its immunogenicity, content of molds, the maximum vigor of maintenance and reach the method that prolongs the cutify survival.

Eight, the diversity ratio of different pig kinds

According to selected optimum condition V3T4S3Z3, to analyze to the stripped Corii Sus domestica of different genera commonly used respectively, different pig kinds are done 3 repetitions, and data are following:

UVB irradiation not of the same race is as shown in table 11 to epiderm skin cytositimulation lymphopoiesis suppression ratio.

Table 11, different pig kind UVB shine epiderm skin cytositimulation lymphopoiesis suppression ratio (%)

The fragrant pig of crust horse	Large White	Rongchang County pig	The Taihu Lake pig
				96.1	91.8	92.8	98.0
89.2	89.7	99.3	95.5
				96.4	94.6	97.3	99.0

Different pig kind UVB irradiation is as shown in table 12 to skin LC quantity decline percentage rate.

Table 12, different pig kind UVB shine skin LC quantity decline percentage rate (%)

The fragrant pig of crust horse	Large White	Rongchang County pig	The Taihu Lake pig
				90.2	88.0	84.6	81.6
82.6	86.5	83.4	80.2
				86.4	80.0	76.7	85.4

Different pig kind UVB are as shown in table 13 to skin content of molds antibacterial decline percentage rate.

Table 13, different pig kind UVB are to skin content of molds antibacterial decline percentage rate (%)

The fragrant pig of crust horse	Large White	Rongchang County pig	The Taihu Lake pig
				96.4	98.1	95.2	84.5
95.7	88.6	82.5	92.4
				89.5	91.4	90.4	91.6

It is as shown in table 14 that the postradiation skin activity percentage rate of different pig kind UVB influences the result.

Table 14, the postradiation skin activity percentage rate of different pig kind UVB influence result (%)

The fragrant pig of crust horse	Large White	Rongchang County pig	The Taihu Lake pig
				74.6	72.1	73.5	64.3
71.7	63.5	70.6	72.5
				67.5	73.2	65.5	75.3

To different pig UVB irradiations back one factor analysis of variance, the result is as shown in Tble 15.

Table 15, different pig kind UVB irradiation back one factor analysis of variance result

Can know by table 15, carry out one factor analysis of variance through SPSS software after, find that significance all greater than 0.05, shows there was no significant difference between the pig kind.Therefore can select the Corii Sus domestica of any pig kind to carry out disinfection, can realize identical goal of the invention by method disclosed by the invention.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Claims (6)

1. utilize ultraviolet B radiation to the isolated skin disinfectant method, it is characterized in that: it is 180 ~ 240V that isolated skin is used voltage, and exposure dose is 100 ~ 400J/cm ²Ultraviolet B radiation irradiation.

According to the said ultraviolet B radiation that utilizes of claim 1 to the isolated skin disinfectant method, it is characterized in that: it is 0 ~ 25 ℃ that said isolated skin places temperature, and humidity is under the environment of 40-70%.

According to the said ultraviolet B radiation that utilizes of claim 2 to the isolated skin disinfectant method, it is characterized in that: it is 25 ℃ that said isolated skin places temperature, and humidity is under 60% environment, uses voltage to be 220V, exposure dose is the ultraviolet B radiation irradiation of 300J/cm.

According to each said ultraviolet B radiation that utilizes of right 1-3 to the isolated skin disinfectant method, it is characterized in that: the ultraviolet B radiation pre-irradiation also comprises the isolated skin pretreatment, is specially isolated skin is processed the material that thickness is 0.8-1.0mm.

According to the said ultraviolet B radiation that utilizes of claim 4 to the isolated skin disinfectant method, it is characterized in that: said isolated skin is for exsomatizing Corii Sus domestica.

6. each said isolated skin that utilizes ultraviolet B radiation to isolated skin disinfectant method preparation of claim 1-5.